Nanoparticles for protein drug delivery

ABSTRACT

The invention discloses a chewable composition for oral delivery of bioactive agents having nanoparticles that are composed of chitosan, poly-glutamic acid, and at least one protein drug or bioactive agent characterized with a positive surface charge and their enhanced permeability for paracellular protein drug and bioactive agent delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/151,230, filed May 5, 2008, which is acontinuation-in-part application of U.S. patent application Ser. No.11/398,145, filed Apr. 5, 2006, now U.S. Pat. No. 7,381,716, which is acontinuation-in-part application of U.S. patent application Ser. No.11/284,734, filed Nov. 21, 2005, now U.S. Pat. No. 7,282,194, which is acontinuation-in-part application of U.S. patent application Ser. No.11/029,082, filed Jan. 4, 2005, now U.S. Pat. No. 7,265,090, the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is related to medical uses of nanoparticles and achewable composition for oral delivery of pharmaceutically-active orbioactive agents having a pharmaceutical composition of chitosan andpolyglutamic acid with bioactive agents and their enhanced permeabilityfor paracellular delivery.

BACKGROUND OF THE INVENTION

Production of pharmaceutically active peptides and proteins in largequantities has become feasible (Biomacromolecules 2004; 5:1917-1925).The oral route is considered the most convenient way of drugadministrations for patients. Nevertheless, the intestinal epithelium isa major barrier to the absorption of hydrophilic drugs such as peptidesand proteins (J. Control. Release 1996; 39:131-138). This is becausehydrophilic drugs cannot easily diffuse across the cells through thelipid bilayer cell membranes. Attentions have been given to improvingparacellular transport of hydrophilic drugs (J. Control. Release 1998;51:35-46). The transport of hydrophilic molecules via the paracellularpathway is, however, severely restricted by the presence of tightjunctions that are located at the luminal aspect of adjacent epithelialcells (Annu. Rev. Nutr. 1995; 15:35-55). These tight junctions form abarrier that limits the paracellular diffusion of hydrophilic molecules.The structure and function of tight junctions is described, inter alia,in Ann. Rev. Physiol. 1998; 60:121-160 and in Ballard T S et al., Annu.Rev. Nutr. 1995; 15:35-55. Tight junctions do not form a rigid barrierbut play an important role in the diffusion through the intestinalepithelium from lumen to bloodstream and vice versa.

Movement of solutes between cells, through the tight junctions that bindcells together into a layer as with the epithelial cells of thegastrointestinal tract, is termed paracellular transport. Paracellulartransport is passive. Paracellular transport depends on electrochemicalgradients generated by transcellular transport and on solvent dragthrough tight junctions. Tight junctions form an intercellular barrierthat separates the apical and basolateral fluid compartments of a celllayer. Movement of a solute through a tight junction from apical tobasolateral compartments depends on the “tightness” of the tightjunction for that solute.

Polymeric nanoparticles have been widely investigated as carriers fordrug delivery (Biomaterials 2002; 23:3193-3201). Much attention has beengiven to the nanoparticles made of synthetic biodegradable polymers suchas poly-ε-caprolactone and polylactide due to their goodbiocompatibility (J. Drug Delivery 2000; 7:215-232; Eur. J. Pharm.Biopharm. 1995; 41:19-25). However, these nanoparticles are not idealcarriers for hydrophilic drugs because of their hydrophobic property.Some aspects of the invention relate to a novel nanoparticle system,composed of hydrophilic chitosan and poly(glutamic acid) hydrogels thatis prepared by a simple ionic-gelation method. This technique ispromising as the nanoparticles are prepared under mild conditionswithout using harmful solvents. It is known that organic solvents maycause degradation of peptide or protein drugs that are unstable andsensitive to their environments (J. Control. Release 2001; 73:279-291).

Following the oral drug delivery route, protein drugs are readilydegraded by the low pH of gastric medium in the stomach. The absorptionof protein drugs following oral administration is challenging due totheir high molecular weight, hydrophilicity, and susceptibility toenzymatic inactivation. Protein drugs at the intestinal epithelium couldnot partition into the hydrophobic membrane and thus can only traversethe epithelial barrier via the paracellular pathway. However, the tightjunction forms a barrier that limits the paracellular diffusion ofhydrophilic molecules.

Chitosan (CS), a cationic polysaccharide, is generally derived fromchitin by alkaline deacetylation (J. Control. Release 2004; 96:285-300).It was reported from literature that CS is non-toxic and soft-tissuecompatible (Biomacromolecules 2004; 5:1917-1925; Biomacromolecules 2004;5:828-833). Additionally, it is known that CS has a special feature ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells (Pharm. Res. 1994; 11:1358-1361).Most commercially available CSs have a quite large molecular weight (MW)and need to be dissolved in an acetic acid solution at a pH value ofapproximately 4.0 or lower that is sometimes impractical. However, thereare potential applications of CS in which a low MW would be essential.Given a low MW, the polycationic characteristic of CS can be usedtogether with a good solubility at a pH value close to physiologicalranges (Eur. J. Pharm. Biopharm. 2004; 57:101-105). Loading of peptideor protein drugs at physiological pH ranges would preserve theirbioactivity. On this basis, a low-MW CS, obtained by depolymerizing acommercially available CS using cellulase, is disclosed herein toprepare nanoparticles of the present invention.

The γ-PGA, an anionic peptide, is a natural compound produced ascapsular substance or as slime by members of the genus Bacillus (Crit.Rev. Biotechnol. 2001; 21:219-232). γ-PGA is unique in that it iscomposed of naturally occurring L-glutamic acid linked together throughamide bonds. It was reported from literature that this naturallyoccurring γ-PGA is a water-soluble, biodegradable, and non-toxicpolymer. A related, but structurally different polymer, [poly(α-glutamicacid), α-PGA] has been used for drug delivery (Adv. Drug Deliver. Rev.2002; 54:695-713; Cancer Res. 1998; 58:2404-2409). α-PGA is usuallysynthesized from poly(γ-benzyl-L-glutamate) by removing the benzylprotecting group with the use of hydrogen bromide. Hashida et al. usedα-PGA as a polymeric backbone and galactose moiety as a ligand to targethepatocytes (J. Control. Release 1999; 62:253-262). Their in vivoresults indicated that the galactosylated α-PGA had a remarkabletargeting ability to hepatocytes and degradation of α-PGA was observedin the liver.

Thanou et al. reported chitosan and its derivatives as intestinalabsorption enhancers (Adv Drug Deliv Rev 2001; 50:S91-S101). Chitosan,when protonated at an acidic pH, is able to increase the paracellularpermeability of peptide drugs across mucosal epithelia.Co-administration of chitosan or trimethyl chitosan chloride withpeptide drugs were found to substantially increase the bioavailabilityof the peptide in animals compared with administrations without thechitosan component.

Oral administration of protein/peptides is advantageous as a drugdelivery route. A novel bioactive nanoparticles system has beendisclosed to effectively deliver the bioactive drugs orally through thegastrointestinal tract. There is a need for preparing chewable productscontaining bioactive nanoparticles that would be safe and efficaciousfor feeding an animal subject, including a human being.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide a novelnanoparticle system and methods of preparation for paracellulartransport drug delivery using a simple and mild ionic-gelation methodupon addition of a poly-γ-glutamic acid (γ-PGA) solution into regularmolecular weight chitosan solution. In one embodiment, the chitosanemployed is N-trimethyl chitosan (TMC). In an alternate embodiment, thechitosan employed is low molecular weight chitosan (low-MW CS). In oneembodiment, the molecular weight of a low-MW CS of the present inventionis about 80 kDa or less, preferably at about 40 kDa, adapted foradequate solubility at a pH that maintains the bioactivity of proteinand peptide drugs. It is stipulated that a chitosan particle with about30-50 kDa molecular weight is kidney inert. The particle size and thezeta potential value of the prepared nanoparticles are controlled bytheir constituted compositions. The results obtained by the TEM(transmission electron microscopy) and AFM (atomic force microscopy)examinations showed that the morphology of the prepared nanoparticleswas generally spherical or spheroidal in shape.

Evaluation of the prepared nanoparticles in enhancing intestinalparacellular transport was investigated in vitro in Caco-2 cellmonolayers. Some aspects of the present invention provide thenanoparticles with CS dominated on the surfaces to effectively reducethe transepithelial electrical resistance (TEER) of Caco-2 cellmonolayers. The confocal laser scanning microscopy (CLSM) observationsconfirm that the nanoparticles with CS dominated on the surface are ableto open the tight junctions between Caco-2 cells and allows transport ofthe nanoparticles via the paracellular pathways.

Some aspects of the invention relate to a method of enhancing intestinalor blood brain paracellular transport configured for delivering at leastone bioactive agent in an animal subject, including an adult patient ora young child, comprising administering nanoparticles composed of γ-PGAand chitosan, wherein the step of administering the nanoparticles may bevia oral administration or injection into a blood vessel. In oneembodiment, the chitosan dominates on a surface of the nanoparticles asshell substrate and the negatively charged γ-PGA as core substrate. Inanother embodiment, a substantial surface of the nanoparticles ischaracterized with a positive surface charge. In a further embodiment,the nanoparticles of the present invention comprise at least onepositively charged shell substrate and at least one negatively chargedcore substrate. In one embodiment, all of the negatively charged coresubstrate conjugates with a portion of the positively charged shellsubstrate that is in the core portion so to maintain a zero-charge(neutral) core. In one embodiment, at least one bioactive or proteindrug is conjugated with the negatively charged core substrate or thezero-charge (neutral) core.

In a further embodiment, the chitosan of the nanoparticles is a lowmolecular weight chitosan, wherein the low molecular weight chitosan hasa molecular weight of about 50 kDa, preferably having a molecular weightof less than about 40 kDa.

In a further embodiment, the nanoparticles have a mean particle sizebetween about 50 and 400 nanometers, preferably between about 100 and300 nanometers, and most preferably between about 100 and 200nanometers.

In some embodiments, the nanoparticles are loaded with a therapeuticallyeffective amount of at least one bioactive agent, wherein the bioactiveagent is selected from the group consisting of proteins, peptides,nucleosides, nucleotides, antiviral agents, antineoplastic agents,antibiotics, and anti-inflammatory drugs.

Further, the bioactive agent may be selected from the group consistingof calcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin,tyrotropin releasing hormone, follicle stimulating hormone, luteinizinghormone, vasopressin and vasopressin analogs, catalase, superoxidedismutase, interleukin-II, interferon, colony stimulating factor, tumornecrosis factor and melanocyte-stimulating hormone. In one preferredembodiment, the bioactive agent is an Alzheimer antagonist.

Some aspects of the invention relate to an oral dose of nanoparticlesthat effectively enhance intestinal or blood brain paracellulartransport comprising γ-PGA or α-PGA and low molecular weight chitosan,wherein the chitosan dominates on a surface of the nanoparticles. Someaspects of the invention relate to an oral dose of nanoparticles thateffectively enhance intestinal or blood brain paracellular transportcomprising a negative component, such as γ-PGA, α-PGA, heparin, orheparan sulfate, in the core and low molecular weight chitosan, whereinthe chitosan dominates on a surface of the nanoparticles with positivecharges.

In a further embodiment, the nanoparticles comprise at least onebioactive agent, such as insulin, insulin analog, Alzheimer's diseaseantagonist, Parkison's disease antagonist, or other protein/peptide. Thebioactive agent for treating Alzheimer's disease may include memantinehydrochloride (Axura® by Merz Pharmaceuticals), donepezil hydrochloride(Aricept® by Eisai Co. Ltd.), rivastigmine tartrate (Exelon® byNovartis), galantamine hydrochloride (Reminyl® by Johnson & Johnson),and tacrine hydrochloride (Cognex® by Parke Davis). Examples of insulinor insulin analog products include, but not limited to, Humulin® (by EliLilly), Humalog® (by Eli Lilly) and Lantus® (by Aventis).

Some aspects of the invention relate to an oral dose of nanoparticlesthat effectively enhance intestinal or blood brain paracellulartransport comprising γ-PGA and low molecular weight chitosan, whereinthe nanoparticles are crosslinked with a crosslinking agent or withlight, such as ultraviolet irradiation.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle. In oneembodiment, the third component is γ-PGA, α-PGA, derivatives or salts ofPGA, heparin or alginate. In another embodiment, the first componentcomprises insulin at a concentration range of 0.075 to 0.091 mg/ml, thesecond component at a concentration range of 0.67 to 0.83 mg/ml, and thethird component comprises γ-PGA at a concentration range of 0.150 to0.184 mg/ml.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein theat least one bioactive agent is an antagonist for Alzheimer's disease oris for treating Alzheimer's disease selected from the group consistingof memantine hydrochloride, donepezil hydrochloride, rivastigminetartrate, galantamine hydrochloride, and tacrine hydrochloride. In afurther embodiment, the at least one bioactive agent is insulin orinsulin analog. In still another embodiment, the at least one bioactiveagent is selected from the group consisting of proteins, peptides,nucleosides, nucleotides, antiviral agents, antineoplastic agents,antibiotics, and anti-inflammatory drugs.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, wherein the nanoparticles are further encapsulated in acapsule or hard-cap capsule. In one embodiment, the nanoparticles arefreeze-dried. In one embodiment, the interior surface of the capsule istreated to be lipophilic or hydrophobic. In another embodiment, theexterior surface of the capsule is enteric-coated.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein thesecond component is crosslinked. In one embodiment, the degree ofcrosslinking is less than 50%. In another embodiment, the degree ofcrosslinking is ranged between 1% and 20%.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein thesecond component is crosslinked with a crosslinking agent selected fromthe group consisting of genipin, its derivatives, analog, stereoisomersand mixtures thereof. In one embodiment, the crosslinking agent isselected from the group consisting of epoxy compounds, dialdehydestarch, glutaraldehyde, formaldehyde, dimethyl suberimidate,carbodiimides, succinimidyls, diisocyanates, acyl azide, reuterin,ultraviolet irradiation, dehydrothermal treatment,tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine andphoto-oxidizers.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, wherein the low molecule weight chitosan has a molecularweight of 80 kDa or less. In one embodiment, the low molecule weightchitosan is further grafted with a polymer.

Some aspects of the invention provide a method of enhancing intestinalor brain blood paracellular transport comprising administering a dose ofnanoparticles, wherein each nanoparticle comprises a first component ofat least one bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle. In oneembodiment, the step of administering the dose of nanoparticles is viaoral administration for enhancing intestinal paracellular transport. Inanother embodiment, the step of administering the dose of nanoparticlesis via venous administration or injection to a blood vessel forenhancing brain blood paracellular transport or reducing the blood-brainbarrier (BBB).

Some aspects of the invention provide a method of treating diabetes ofan animal subject, including a human being, comprising orallyadministering insulin containing nanoparticles with a dosage effectiveamount of the insulin to treat the diabetes, wherein at least a portionof the nanoparticles comprises a positively charged shell substrate anda negatively charged core substrate. In one embodiment, the shellsubstrate comprises chitosan, chitin, chitosan oligosaccharides, andchitosan derivatives thereof, wherein a substantial portion of a surfaceof the nanoparticles is characterized with a positive surface charge. Inanother embodiment, the core substrate is selected from the groupconsisting of γ-PGA, α-PGA, water-soluble salts of PGA, metal salts ofPGA, heparin, heparin analogs, low molecular weight heparin,glycosaminoglycans, and alginate. The molecular formula of the insulinis selected from the group consisting of C₂₅₄H₃₇₇N₆₅O₇₅S₆,C₂₅₇H₃₈₃N₆₅O₇₇S₆, C₂₅₆H₃₈₁N₆₅O₇₉S₆, C₂₆₇H₄₀₄N₇₂O₇₈S₆, C₂₆₇H₄₀₈N₇₂O₇₇S₆(insulin glargine), C₂₆₇H₄₀₂N₆₄O₇₆S₆ (insulin determir), and the like.

In one embodiment, the orally administering insulin containingnanoparticles comprise a dosage effective amount of the insulin to treatthe diabetes comprising an insulin amount of between about 15 units to45 units, preferably between about 25 units to 35 units, per kilogrambody weight of the animal subject. In a further embodiment, theinsulin-containing nanoparticle comprises a trace amount of zinc orcalcium, or is treated with enteric coating.

In one embodiment, the insulin containing nanoparticles further compriseat least one paracellular transport enhancer, wherein the paracellulartransport enhancer may be selected from the group consisting of Ca²⁺chelators, bile salts, anionic surfactants, medium-chain fatty acids,and phosphate esters. In another embodiment, the nanoparticles and theparacellular transport enhancer are co-encapsulated in a capsule or areencapsulated separately.

Some aspects of the invention provide nanoparticles for oraladministration in an animal subject, including a patient, comprising apositively charged shell substrate, a negatively charged core substrate,and a bioactive agent conjugated with the core substrate, wherein thecore substrate is selected from the group consisting of heparin, heparinanalogs, low molecular weight heparin, glycosaminoglycans, and alginate,the bioactive agent being selected from the group consisting ofchondroitin sulfate, hyaluronic acid, growth factor and protein withpharmaceutically effective amount.

Some aspects of the invention provide nanoparticles for oraladministration in an animal subject, including a patient, comprising apositively charged shell substrate, a negatively charged core substrate,and a bioactive agent conjugated with the core substrate, wherein thebioactive agent is calcitonin or vancomycin.

Some aspects of the invention provide a method of treating Alzheimer'sdiseases of an animal subject, including a patient, comprisingintravenously administering bioactive nanoparticles with a dosageeffective to treat the Alzheimer's diseases, wherein the bioactivenanoparticles comprises a positively charged shell substrate, anegatively charged core substrate, and at least one bioactive agent fortreating Alzheimer's disease, wherein the at least one bioactive agentis selected from the group consisting of memantine hydrochloride,donepezil hydrochloride, rivastigmine tartrate, galantaminehydrochloride, and tacrine hydrochloride.

In one embodiment, the dosage effective to treat the Alzheimer'sdiseases comprises administering the at least one bioactive agent fortreating Alzheimer's disease at about 10 mg to 40 mg per day over aperiod of one month to one year. In another embodiment, at least aportion the shell substrate is crosslinked, preferably at a degree ofcrosslinking less than about 50%, or most preferably between about 1%and 20%.

One aspect of the invention provides a pharmaceutical composition ofnanoparticles, wherein the nanoparticles may be freeze-dried to formsolid dried nanoparticles. The dried nanoparticles may be loaded in acapsule (such as a two-part hard gelatin capsule) for oraladministration in an animal subject, including a patient, wherein thecapsule may be further enterically coated. The freeze-driednanoparticles can be rehydrated in solution or by contacting fluid so torevert to wet nanoparticles having positive surface charge. In oneembodiment, nanoparticles may be mixed with trehalose or withhexan-1,2,3,4,5,6-hexyl in a freeze-drying process. In one embodiment,the interior surface of the capsule is treated to be lipophilic orhydrophobic. In another embodiment, the exterior surface of the capsuleis enteric-coated.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles characterized by enhancing paracellular transport, eachnanoparticle comprising a shell component and a core component, whereinat least a portion of the shell component comprises chitosan and whereinthe core component is comprised of MgSO₄, sodium tripolyphosphate, atleast one bioactive agent, and a negatively charged compound, wherein asubstantial portion of the negatively charged compound is conjugated tothe chitosan. In one embodiment, the negatively charged component of thepharmaceutical composition is γ-PGA or a derivative or salt of PGAs.

Some aspects of the invention provide an orally deliverable capsule toan animal subject comprising: (a) an empty capsule; and (b) bioactivenanoparticles loaded within the empty capsule, wherein the nanoparticlescomprise a shell substrate of chitosan, a negatively charged coresubstrate, and at least one bioactive agent. In one embodiment, theempty capsule comprises a two-part hard gelatin capsule. In anotherembodiment, the capsule is treated with enteric coating.

One object of the present invention is to provide a method ofmanufacturing the orally deliverable capsule, the method comprisingsteps of: (a) providing an empty capsule; (b) providing bioactivenanoparticles, wherein the nanoparticles comprise a shell substrate ofchitosan, a negatively charged core substrate, and at least onebioactive agent; (c) freeze-drying the nanoparticles; and (d) fillingthe freeze-dried bioactive nanoparticles into the empty capsule, therebyproducing an orally deliverable capsule. In one embodiment, thebioactive nanoparticles further comprise magnesium sulfate and TPP.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles for oral administration in an animal subject, including apatient, the nanoparticles comprising a shell portion that is dominatedby positively charged chitosan, a core portion that contains negativelycharged substrate, wherein the negatively charged substrate is at leastpartially neutralized with a portion of the positively charged chitosanin the core portion, and at least one bioactive agent loaded within thenanoparticles. In one embodiment, the bioactive agent is a non-insulinexenatide, a non-insulin pramlintide, insulin, insulin analog, orcombinations thereof. In one embodiment, the nanoparticles are formedvia a simple and mild ionic-gelation method.

In one embodiment of the pharmaceutical composition of the presentinvention, the substrate is PGA, wherein the PGA may be γ-PGA, α-PGA,PGA derivatives, or salts of PGA. In one embodiment of thepharmaceutical composition of the present invention, the substrate isheparin, wherein the heparin is a low molecular weight heparin.

In one embodiment, a surface of the nanoparticles of the pharmaceuticalcomposition of the present invention is characterized with a positivesurface charge, wherein the nanoparticles have a surface charge fromabout +15 mV to about +50 mV. In another embodiment, the nanoparticleshave a mean particle size between about 50 and 400 nanometers. In stillanother embodiment, at least a portion of the shell portion of thenanoparticles is crosslinked. In a further embodiment, the nanoparticlesare in a form of freeze-fried powder. In one embodiment, thenanoparticles of the pharmaceutical composition of the present inventionfurther comprise magnesium sulfate and TPP.

In one embodiment, the nanoparticles of the pharmaceutical compositionof the present invention are encapsulated in a capsule, wherein anexterior surface of the capsule may be treated with enteric coating andan interior surface of the capsule may be treated with hydrophobiccoating.

In one embodiment, the chitosan has a molecular weight about 80 kDa orless. In another embodiment, the chitosan is trimethyl chitosan.

Some aspects of the invention provide a method of delivering a bioactiveagent to blood circulation in an animal subject, including a patient,comprising: (a) providing nanoparticles according to the pharmaceuticalcomposition of the present invention, wherein the nanoparticles areformed via a simple and mild ionic-gelation method; (b) administeringthe nanoparticles orally toward an intestine of the animal subject,including a patient; (c) urging the nanoparticles to be absorbed onto asurface of an epithelial membrane of the intestine; (d) permeatingbioactive agent to pass through an epithelial barrier of the intestine;and (e) releasing the bioactive agent into the blood circulation. In oneembodiment, the bioactive agent is selected from the group consisting ofexenatide, pramlintide, insulin, insulin analog, and combinationsthereof.

Some aspects of the invention provide a chewable composition as animalfood for feeding an animal subject comprising a filler substance andnanoparticles that are blended with (or mixed in) the filler substance,the nanoparticles comprising a shell portion that is dominated bypositively charged chitosan, a core portion that contains negativelycharged substrate, wherein the negatively charged substrate is at leastpartially neutralized with a portion of the positively charged chitosanin the core portion, and at least one bioactive agent loaded within thenanoparticles. In one embodiment, the chewable (or chewable animal food)is configured for oral administration in an animal subject, includinghuman being. In another embodiment, the chewable animal food in is theform of tablets, soft chewable tablets, pills, or other appropriateconfigurations.

In one embodiment, the substrate of the chewable composition in the coreportion is PGA or heparin. In another embodiment, the nanoparticles inthe chewable composition are formed via a simple and mild ionic-gelationmethod. In still another embodiment, the nanoparticles of the chewablecomposition further comprise magnesium sulfate and TPP in the coreportion.

In a preferred embodiment, the chitosan of the nanoparticles of thechewable composition is trimethyl chitosan. In another embodiment, thebioactive agent of the chewable composition is insulin or insulinanalog.

In one embodiment, the nanoparticles are freeze-fried before beingblended with the filler substance. In another embodiment, the bioactiveagent of the chewable composition is exenatide or pramlintide. In stillanother embodiment, the bioactive agent of the chewable composition isprotein or peptide.

In one embodiment, the nanoparticles of the chewable composition areembedded in a jerry. In another embodiment, the bioactive agent of thechewable composition is calcitonin or vancomycin.

In one embodiment, the nanoparticles of the chewable composition arecoated with hydrogels. In another embodiment, the filler substance ofthe chewable composition comprises excipients. In still anotherembodiment, the bioactive agent of the chewable composition is hormoneor growth hormone.

In one embodiment, the nanoparticles of the chewable composition areencapsulated in or coated with an alginate-calcium matrix. In oneembodiment, the nanoparticles of the chewable composition furthercomprise an absorption enhancer disclosed herein. In another embodiment,the nanoparticles of the chewable composition are loaded in organogelsor xerogels.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional objects and features of the present invention will becomemore apparent and the disclosure itself will be best understood from thefollowing Detailed Description of the Exemplary Embodiments, when readwith reference to the accompanying drawings.

FIG. 1 shows GPC chromatograms of (a) standard-MW CS beforedepolymerization and the low-MW CS after depolymerization; (b) thepurified γ-PGA obtained from microbial fermentation.

FIG. 2 shows (a) FT-IR and (b) ¹H-NMR spectra of the purified γ-PGAobtained from microbial fermentation.

FIG. 3 shows FT-IR spectra of the low-MW CS and the prepared CS-γ-PGAnanoparticles.

FIG. 4 shows (a) a TEM micrograph of the prepared CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) an AFM micrograph of the preparedCS-7-PGA nanoparticles (0.01% γ-PGA:0.01% CS).

FIG. 5 shows changes in particle size and zeta potential of (a) theCS-γ-PGA nanoparticles (0.10% γ-PGA:0.20% CS) and (b) the CS-7-PGAnanoparticles (0.10% γ-PGA:0.01% CS) during storage for up to 6 weeks.

FIG. 6 shows effects of the prepared CS-γ-PGA nanoparticles on the TEERvalues of Caco-2 cell monolayers.

FIG. 7 shows fluorescence images (taken by an inversed confocal laserscanning microscope) of 4 optical sections of a Caco-2 cell monolayerthat had been incubated with the fCS-γ-PGA nanoparticles with a positivesurface charge (0.10% γ-PGA:0.20% CS) for (a) 20 min and (b) 60 min.

FIG. 8 shows an illustrative protein transport mechanism through a celllayer, including transcellular transport and paracelluler transport.

FIG. 9 shows a schematic illustration of a paracellular transportmechanism.

FIG. 10 shows an fCS-γ-PGA nanoparticle with FITC-labeled chitosanshaving positive surface charge.

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA.

FIG. 12 shows loading capacity and association efficiency of insulin innanoparticles of chitosan as reference.

FIG. 13 shows the stability of insulin-loaded nanoparticles.

FIG. 14 shows a representative in vitro study with insulin drug releaseprofile in a pH-adjusted solution.

FIG. 15 shows the effect of insulin of orally administeredinsulin-loaded nanoparticles on hypoglycemia in diabetic rats.

FIG. 16 shows a proposed mechanism of nanoparticles released from theenteric coated capsules.

FIG. 17 shows the schematic illustration of insulin conjugated withhistidine and/or glutamic acid side groups of the γ-PGA via zinc.

FIG. 18 shows the schematic illustration of insulin conjugated with acarboxyl side group of the γ-PGA via zinc.

FIG. 19 shows the effect of orally administered insulin-loadednanoparticles on ‘glucose reduction %’ in diabetic rats, wherein thefreeze-dried nanoparticles were loaded in an enterically coated capsuleupon delivery.

FIG. 20 shows insulin-loaded nanoparticles with a core compositionconsisted of γ-PGA, MgSO₄, sodium tripolyphosphate (TPP), and insulin.

FIG. 21 shows an in vivo subcutaneous study using insulin injectablesand insulin-containing nanoparticles.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The preferred embodiments of the present invention described belowrelate particularly to preparation of nanoparticles composed ofchitosan/poly-glutamic acid/insulin and their permeability to enhancethe intestinal or blood brain paracellular permeation by opening thetight junctions between epithelial cells. While the description setsforth various embodiment specific details, it will be appreciated thatthe description is illustrative only and should not be construed in anyway as limiting the invention. Furthermore, various applications of theinvention, and modifications' thereto, which may occur to those who areskilled in the art, are also encompassed by the general conceptsdescribed below.

γ-PGA is a naturally occurring anionic homo-polyamide that is made ofL-glutamic acid units connected by amide linkages between α-amino andγ-carboxylic acid groups (Crit. Rev. Biotechnol. 2001; 21:219-232). Itis an exocellular polymer of certain Bacillus species that is producedwithin cells via the TCA cycle and is freely excreted into thefermentation broth. Its exact biological role is not fully known,although it is likely that γ-PGA is linked to increasing the survival ofproducing strains when exposed to environmental stresses. Because of itswater-solubility, biodegradability, edibility, and non-toxicity towardhumans and the environment, several applications of γ-PGA in food,cosmetics, medicine, and water treatment have been investigated in thepast few years.

Example No. 1 Materials and Methods of Nanoparticles Preparation

CS (MW ˜2.8×10⁵) with a degree of deacetylation of approximately 85% wasacquired from Challenge Bioproducts Co. (Taichung, Taiwan). Acetic acid,cellulase (1.92 units/mg), fluorescein isothiocyanate (FITC), phosphatebuffered saline (PBS), periodic acid, sodium acetate, formaldehyde,bismuth subnitrate, and Hanks balanced salt solution (HBSS) werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Ethanol absoluteanhydrous and potassium sodium tartrate were obtained from Merck(Darmstadt, Germany). Non-essential amino acid (NEAA) solution, fetalbovine serum (FBS), gentamicin and trypsin-EDTA were acquired from Gibco(Grand Island, N.Y.). Eagle's minimal essential medium (MEM) waspurchased from Bio West (Nuaille, France). All other chemicals andreagents used were of analytical grade.

Example No. 2 Depolymerization of CS by Enzymatic Hydrolysis

Regular CS was treated with enzyme (cellulase) to produce low-MW CSaccording to a method described by Qin et al. with some modifications(Food Chem. 2004; 84:107-115). A solution of CS (20 g/l) was prepared bydissolving CS in 2% acetic acid. Care was taken to ensure totalsolubility of CS. Then, the CS solution was introduced into a vessel andadjusted to the desired pH 5.0 with 2N aqueous NaOH. Subsequently,cellulase (0.1 g) was added into the CS solution (100 ml) andcontinuously stirred at 37° C. for 12 hours. Afterward, thedepolymerized CS was precipitated with aqueous NaOH at pH 7.0-7.2 andthe precipitated CS was washed three times with deionized water. Theresulting low-MW CS was lyophilized in a freeze dryer (Eyela Co. Ltd,Tokyo, Japan).

The average molecular weight of the depolymerized CS was determined by agel permeation chromatography (GPC) system equipped with a series of PLaquagel-OH columns (one Guard 8 μm, 50×7.5 mm and two MIXED 8 μm,300×7.5 mm, PL Laboratories, UK) and a refractive index (R1) detector(RI2000-F, SFD, Torrance, Calif.). Polysaccharide standards (molecularweights range from 180 to 788,000, Polymer Laboratories, UK) were usedto construct a calibration curve. The mobile phase contained 0.01MNaH₂PO₄ and 0.5M NaNO₃ and was brought to a pH of 2.0. The flow rate ofmobile phase was 1.0 ml/min, and the columns and the R1 detector cellwere maintained at 30° C.

Factors limiting applications of most commercially available CSs aretheir high molecular weight and thus high viscosity and poor solubilityat physiological pH ranges. Low-MW CS overcomes these limitations andhence finds much wider applications in diversified fields. It wassuggested that low-MW CS be used as a parenteral drug carrier due to itslower antigen effect (Eur. J. Pharm. Biopharm. 2004; 57:101-105). Low-MWCS was used as a non-viral gene delivery system and showed promisingresults (Int. J. Pharm. 1999; 178:231-243). Other studies based onanimal testing showed the possibilities of low-MW CS for treatment oftype 2 diabetes and gastric ulcer (Biol. Pharm. Bull. 2002; 25:188-192).Several hydrolytic enzymes such as lysozyme, pectinase, cellulase,bromelain, hemicellulase, lipase, papain and the like can be used todepolymerize CS (Biochim. Biophys. Acta 1996; 1291:5-15; Biochem. Eng.J. 2001; 7:85-88; Carbohydr. Res. 1992; 237:325-332).

FIG. 1 a shows GPC chromatograms of both standard-MW (also known asregular-MW) and low-MW CS. It is known that cellulase catalyzes thecleavage of the glycosidic linkage in CS (Food Chem. 2004; 84:107-115).The low-MW CS used in the study was obtained by precipitating thedepolymerized CS solution with aqueous NaOH at pH 7.0-7.2. Thus,obtained low-MW CS had a MW of about 50 kDa (FIG. 1 a). In a preferredembodiment, the low molecular weight chitosan has a molecular weight ofless than about 40 kDa, but above 10 kDa. Other forms of chitosan mayalso be applicable, including chitin, chitosan oligosaccharides, andderivatives thereof.

It was observed that the obtained low-MW CS can be readily dissolved inan aqueous solution at pH 6.0, while that before depolymerization needsto be dissolved in an acetic acid solution with a pH value about 4.0.Additionally, it was found that with the low-MW CS, the preparednanoparticles had a significantly smaller size with a narrowerdistribution than their counterparts prepared with the high-MW (alsoknown as standard-MW) CS (before depolymerization), due to its lowerviscosity. As an example, upon adding a 0.10% γ-PGA aqueous solutioninto a 0.20% high-MW CS solution (viscosity 5.73±0.08 cp, measured by aviscometer), the mean particle size of the prepared nanoparticles was878.3±28.4 nm with a polydispersity index of 1.0, whereas adding a 0.10%γ-PGA aqueous solution into the low-MW CS solution (viscosity 1.29±0.02cp) formed nanoparticles with a mean particle size of 218.1±4.1 nm witha polydispersity index of 0.3 (n=5).

Example No. 3 Production and Purification of γ-PGA

γ-PGA was produced by Bacillus licheniformis (ATCC 9945, BioresourcesCollection and Research Center, Hsinchu, Taiwan) as per a methodreported by Yoon et al. with slight modifications (Biotechnol. Lett.2000; 22:585-588). Highly mucoid colonies (ATCC 9945a) were selectedfrom Bacillus licheniformis (ATCC 9945) cultured on the E medium(ingredients comprising L-glutamic acid, 20.0 g/l; citric acid, 12.0g/l; glycerol, 80.0 g/l; NH₄Cl, 7.0 g/l; K₂HPO₄, 0.5 g/l; MgSO₄.7H₂O,0.5 g/l; FeCl₃.6H₂O, 0.04 g/l; CaCl₂-2H₂O, 0.15 g/l; MnSO₄.H₂O, 0.104g/l, pH 6.5) agar plates at 37° C. for several times. Subsequently,young mucoid colonies were transferred into 10 ml E medium and grown at37° C. in a shaking incubator at 250 rpm for 24 hours. Afterward, 500 μlof culture broth was mixed with 50 ml E medium and was transferred intoa 2.5-1 jar-fermentor (KMJ-2B, Mituwa Co., Osaka, Japan) containing 950ml of E medium. Cells were cultured at 37° C. The pH was controlled at6.5 by automatic feeding of 25% (v/v) NH₄OH and/or 2M HCl. The dissolvedoxygen concentration was initially controlled at 40% of air saturationby supplying air and by controlling the agitation speed up to 1000 rpm.

After 40 hours, cells were separated from the culture broth bycentrifugation for 20 minutes at 12,000×g at 4° C. The supernatantcontaining γ-PGA was poured into 4 volumes of methanol and leftovernight with gentle stirring. The resulting precipitate containingcrude γ-PGA was collected by centrifugation for 40 minutes at 12,000×gat 4° C. and then was dissolved in deionized water to remove insolubleimpurities by centrifugation for 20 minutes at 24,000×g at 4° C. Theaqueous γ-PGA solution was desalted by dialysis (MWCO: 100,000, SpectrumLaboratories, Inc., Laguna Hills, Calif.) against distilled water for 12hours with water exchanges several times, and finally was lyophilized toobtain pure γ-PGA.

The purified γ-PGA was verified by the proton nuclear magnetic resonance(¹H-NMR) and the FT-IR analyses. Analysis of ¹H-NMR was conducted on anNMR spectrometer (Varian Unityionva 500 NMR Spectrometer, MO) usingDMSO-d₆ at 2.49 ppm as an internal reference. Test samples used for theFT-IR analysis first were dried and ground into a powder form. Thepowder then was mixed with KBr (1:100) and pressed into a disk. Analysiswas performed on an FT-IR spectrometer (Perkin Elmer Spectrum RX1 FT-IRSystem, Buckinghamshire, England). The samples were scanned from400-4000 cm⁻¹. The average molecular weight of the purified γ-PGA wasdetermined by the same GPC system as described before. Polyethyleneglycol (molecular weights of 106-22,000) and polyethylene oxide(molecular weights of 20,000-1,000,000, PL Laboratories) standards wereused to construct a calibration curve. The mobile phase contained 0.01MNaH₂PO₄ and 0.2M NaNO₃ and was brought to a pH of 7.0.

The purified γ-PGA obtained from fermentation was analyzed by GPC,¹H-NMR, and FT-IR. As analyzed by GPC (FIG. 1 b), the purified γ-PGA hada MW of about 160 kDa. In the FT-IR spectrum (FIG. 2 a), acharacteristic peak at 1615 cm⁻¹ for the associated carboxylic acid salt(—COO⁻ antisymmetric stretch) on γ-PGA was observed. The characteristicabsorption due to C═O in secondary amides (amide I band) was overlappedby the characteristic peak of —COO⁻. Additionally, the characteristicpeak observed at 3400 cm⁻¹ was the N—H stretch of γ-PGA. In the ¹H-NMRspectrum (FIG. 2 b), six chief signals were observed at 1.73 and 1.94ppm (β-CH₂), 2.19 ppm (γ-CH₂), 4.14 ppm (α-CH), 8.15 ppm (amide), and12.58 ppm (COOH). These results indicated that the observed FT-IR and¹H-NMR spectra correspond well to those expected for γ-PGA.Additionally, the fermented product after purification showed nodetected macromolecular impurities by the ¹H-NMR analysis, suggestingthat the obtained white power of γ-PGA is highly pure.

Example No. 4 Preparation of the CS-γ-PGA Nanoparticles

Nanoparticles were obtained upon addition of γ-PGA aqueous solution. (pH7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTechScientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10ml) at varying concentrations (0.01%, 0.05%, 0.10%, 0.15%, or 0.20% byw/v) under magnetic stirring at room temperature. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Supernatantswere discarded and nanoparticles were resuspended in deionized water forfurther studies. FT-IR was used to analyze peak variations of aminogroups of low-MW CS and carboxylic acid salts of γ-PGA in the CS-γ-PGAnanoparticles.

As stated, nanoparticles were obtained instantaneously upon addition ofa γ-PGA aqueous solution (pH 7.4) into a low-MW CS aqueous solution (pH6.0) under magnetic stirring at room temperature. FIG. 3 shows the FT-IRspectra of the low-MW CS and the CS-γ-PGA nanoparticles. As shown in thespectrum of CS, the characteristic peak observed at 1563 cm⁻¹ was theprotonated amino group (—NH₃ ⁺ deformation) on CS. In the spectrum ofCS-γ-PGA complex, the characteristic peak at 1615 cm⁻¹ for —COO⁻ onγ-PGA disappeared and a new peak at 1586 cm⁻¹ appeared, while thecharacteristic peak of —NH₃ ⁺ deformation on CS at 1563 cm⁻¹ shifted to1555 cm⁻¹. These observations are attributed to the electrostaticinteraction between the negatively charged carboxylic acid salts (—COO⁻)on γ-PGA and the positively charged amino groups (—NH₃ ⁺) on CS (Int. J.Pharm. 2003; 250:215-226). The electrostatic interaction between the twopolyelectrolytes (γ-PGA and CS) instantaneously induced the formation oflong hydrophobic segments (or at least segments with a high density ofneutral ion-pairs), and thus resulted in highly neutralized complexesthat segregated into colloidal nanoparticles (Langmuir. 2004;20:7766-7778).

Example No. 5 Characterization of the CS-γ-PGA nanoparticles

The morphological examination of the CS-γ-PGA nanoparticles wasperformed by TEM (transmission electron microscopy) and AFM (atomicforce microscopy). The TEM sample was prepared by placing a drop of thenanoparticle solution onto a 400 mesh copper grid coated with carbon.About 2 minutes after deposition, the grid was tapped with a filterpaper to remove surface water and positively stained by using analkaline bismuth solution (Microbiol. Immunol. 1986; 30:1207-1211). TheAFM sample was prepared by casting a drop of the nanoparticle solutionon a slide glass and then dried in vacuum. The size distribution andzeta potential of the prepared nanoparticles were measured using aZetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK).

During storage, aggregation of nanoparticles may occur and thus leads tolosing their structural integrity or forming precipitation ofnanoparticles (Eur. J. Pharm. Sci. 1999; 8:99-107). Therefore, thestability of nanoparticles during storage must be evaluated. In thestability study, the prepared nanoparticles suspended in deionized water(1 mg/ml) were stored at 4° C. and their particle sizes and zetapotential values were monitored by the same Zetasizer as mentionedearlier during storage.

In the preparation of nanoparticles, samples were visually analyzed andthree distinct solution systems were identified: clear solution,opalescent suspension, and solution with precipitation of aggregates.Examined by the Zetasizer, nanoparticles were found in the clearsolution and the opalescent suspension rather than in the solution withprecipitation of aggregates.

The particle sizes and the zeta potential values of CS-γ-PGAnanoparticles, prepared at varying concentrations of γ-PGA and CS, weredetermined and the results are shown in Tables 1a and 1b. It was foundthat the particle size and the zeta potential value of the preparednanoparticles were mainly determined by the relative amount of the localconcentration of γ-PGA in the added solution to the surroundingconcentration of CS in the sink solution. At a fixed concentration ofCS, an increase in the γ-PGA concentration allowed γ-PGA moleculesinteracting with more CS molecules, and thus formed a lager size ofnanoparticles (Table 1a, p<0.05). When the amount of CS moleculesexceeded that of local γ-PGA molecules, some of the excessive CSmolecules were entangled onto the surfaces of CS-γ-PGA nanoparticles.

Thus, the resulting nanoparticles may display a structure of a neutralpolyelectrolyte-complex core surrounded by a positively charged CS shell(Table 1b) ensuring the colloidal stabilization (Langmuir. 2004;20:7766-7778). In contrast, as the amount of local γ-PGA moleculessufficiently exceeded that of surrounding CS molecules, the formednanoparticles had γ-PGA exposed on the surfaces and thus had a negativecharge of zeta potential. Therefore, the particle size and the zetapotential value of the prepared CS-γ-PGA nanoparticles can be controlledby their constituted compositions. The results obtained by the TEM andAFM examinations showed that the morphology of the preparednanoparticles was spherical in shape with a smooth surface (FIGS. 4 aand 4 b). Some aspects of the invention relate to nanoparticles having amean particle size between about 50 and 400 nanometers, preferablybetween about 100 and 300 nanometers, and most preferably between about100 and 200 nanometers. The morphology of the nanoparticles showsspherical in shape with a smooth surface at any pH between 2.5 and 6.6.In one embodiment, the stability of the nanoparticles of the presentinvention at a low pH around 2.5 enables the nanoparticles to be intactwhen exposed to the acidic medium in the stomach.

Two representative groups of the prepared nanoparticles were selectedfor the stability study: one with a positive surface charge (0.10%γ-PGA:0.20% CS) and the other with a negative surface charge (0.10%γ-PGA:0.01% CS). FIG. 5 shows changes in particle size (▪, meandiameter) and zeta potential () of (a) the CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) the CS-γ-PGA nanoparticles (0.10%γ-PGA:0.01% CS) during storage for up to 6 weeks. It was found thatneither aggregation nor precipitation of nanoparticles was observedduring storage for up to 6 weeks, as a result of the electrostaticrepulsion between the positively charged CS-γ-PGA nanoparticles (for theformer group) or the negatively charged CS-γ-PGA nanoparticles (for thelatter group).

Additionally, changes in particle size and zeta potential of thenanoparticles were minimal for both studied groups (FIGS. 5 a and 5 b).These results demonstrated that the prepared nanoparticles suspended indeionized water were stable during storage.

TABLE 1a Effects of concentrations of γ-PGA and CS on the particle sizesof the prepared CS-γ-PGA nanoparticles Mean Particle Size (nm, n = 5) CSγ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%^(b))  79.0 ± 3.0 103.1 ±4.6  96.7 ± 1.9 103.6 ± 1.9 140.5 ± 2.0 0.05% 157.4 ± 1.7 120.8 ± 3.9144.5 ± 2.4 106.2 ± 3.8 165.4 ± 1.7 0.10% 202.2 ± 3.1 232.6 ± 1.2 161.0± 1.8 143.7 ± 2.7 218.1 ± 4.1 0.15% 277.7 ± 3.2 264.9 ± 2.1 188.6 ± 2.9178.0 ± 2.2 301.1 ± 6.4 0.20% 284.1 ± 2.1 402.2 ± 4.0 ▴ 225.5 ± 3.1365.5 ± 5.1 ^(a))concentration of CS (by w/v) ^(b))concentration ofγ-PGA (by w/v) ▴ precipitation of aggregates was observed

TABLE 1b Effects of concentrations of γ-PGA and CS on the zeta potentialvalues of the prepared CS-γ-PGA nanoparticles. Zeta Potential (mV, n =5) CS γ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%^(b))   15.4 ± 0.322.8 ± 0.5 19.8 ± 1.5 16.5 ± 1.4 17.2 ± 1.6 0.05% −32.7 ± 0.7 23.7 ± 1.727.6 ± 0.7 20.3 ± 0.8 19.2 ± 0.6 0.10% −33.1 ± 1.3 21.1 ± 1.6 20.3 ± 1.123.6 ± 0.9 24.7 ± 1.2 0.15% −33.2 ± 2.1 −21.9 ± 2.0   19.2 ± 0.4 16.9 ±1.7 19.8 ± 0.3 0.20% −34.5 ± 0.5 −34.6 ± 0.3   ▴ 14.6 ± 0.7 16.3 ± 0.7^(a))concentration of CS (by w/v) ^(b))concentration of γ-PGA (by w/v) ▴precipitation of aggregates was observed

In a further study, NPs were self-assembled instantaneously uponaddition of an aqueous γ-PGA into an aqueous TMC (N-trimethyl chitosan)having a TMC/γ-PGA weight ratio of 6:1 under magnetic stirring at roomtemperature. The chemical formulas of chitosan and N-trimethyl chitosanare shown below:

The amount of positively charged TMC significantly exceeded that ofnegatively charged γ-PGA; some of excessive TMC molecules were entangledonto the surfaces of NPs, thus displaying a positive surface charge(Table 2). The degree of quaternization on TMC had little effects on themean particle size and zeta potential of NPs.

TABLE 2 Mean particle sizes, zeta potential values and polydispersityindices of nanoparticles (NPs) self-assembled by TMC polymers withdifferent degrees of quaternization and γ-PGA (n = 5 batches). MeanParticle  Zeta Potential Polydispersity Size (nm) (mV) Index CS/γ-PGANPs 104.1 ± 1.2 36.2 ± 2.5 0.11 ± 0.02 TMC25/γ-PGA NPs 101.3 ± 3.1 30.9± 2.1 0.13 ± 0.04 TMC40/γ-PGA NPs 106.3 ± 2.3 32.3 ± 2.1 0.15 ± 0.14TMC55/γ-PGA NPs 114.6 ± 2.3 30.6 ± 3.8 0.12 ± 0.03 TMC: N-trimethylchitosan; CS: chitosan; γ-PGA: poly(γ-glutamic acid).

Example No. 6 Caco-2 Cell Cultures and TEER Measurements

Caco-2 cells were seeded on the tissue-culture-treated polycarbonatefilters (diameter 24.5 mm, growth area 4.7 cm²) in Costar Transwell 6wells/plates (Corning Costar Corp., NY) at a seeding density of 3×10⁵cells/insert. MEM (pH 7.4) supplemented with 20% FBS, 1% NEAA, and 40μg/ml antibiotic-gentamicin was used as the culture medium, and added toboth the donor and acceptor compartments. The medium was replaced every48 hours for the first 6 days and every 24 hours thereafter. Thecultures were kept in an atmosphere of 95% air and 5% CO₂ at 37° C. andwere used for the paracellular transport experiments 18-21 days afterseeding (TEER values in the range of 600-800 Ωcm²).

TEER values of the Caco-2 cell monolayers were monitored with aMillicell®-Electrical Resistance System (Millipore Corp., Bedford,Mass.) connected to a pair of chopstick electrodes. To initiate thetransport experiments, the culture media in the donor and acceptorcompartments were aspirated, and the cells were rinsed twice withpre-warmed transport media (HBSS supplemented with 25 mM glucose, pH6.0). Following a 30-min equilibration with the transport media at 37°C., the cells were incubated for 2 hours with 2 ml transport mediacontaining 0.5 ml test nanoparticle solutions (0.2 mg/ml) at 37° C.Subsequently, solutions of nanoparticles were carefully removed andcells were washed three times with HBSS and replaced by fresh culturemedia. The TEER was measured for another 20 hours to study reversibilityof the effect of test nanoparticles on Caco-2 cell monolayers (Eur. J.Pharm. Sci. 2000; 10:205-214).

The intercellular tight junction is one of the major barriers to theparacellular transport of macromolecules (J. Control. Release 1996;39:131-138; J. Control. Release 1998; 51:35-46). Trans-epithelial iontransport is contemplated to be a good indication of the tightness ofthe junctions between cells and was evaluated by measuring TEER ofCaco-2 cell monolayers in the study. It was reported that themeasurement of TEER could be used to predict the paracellular transportof hydrophilic molecules (Eur. J. Pharm. Biopharm. 2004; 58:225-235).When the tight junctions open, the TEER value will be reduced due to thewater and ion passage through the paracellular route. Caco-2 cellmonolayers have been widely used as an in vitro model to evaluate theintestinal paracellular permeability of macromolecules.

Effects of the prepared CS-γ-PGA nanoparticles on the TEER values ofCaco-2 cell monolayers are shown in FIG. 6. As shown, the preparednanoparticles with a positive surface charge (CS dominated on thesurface, 0.01% γ-PGA:0.05% CS, 0.10% γ-PGA:0.2% CS, and 0.20%γ-PGA:0.20% CS) were able to reduce the values of TEER of Caco-2 cellmonolayers significantly (p<0.05). After a 2-hour incubation with thesenanoparticles, the TEER values of Caco-2 cell monolayers were reduced toabout 50% of their initial values as compared to the control group(without addition of nanoparticles in the transport media). Thisindicated that the nanoparticles with CS dominated on the surfaces couldeffectively open or loosen the tight junctions between Caco-2 cells,resulting in a decrease in the TEER values. It was reported thatinteraction of the positively charged amino groups of CS with thenegatively charged sites on cell surfaces and tight junctions induces aredistribution of F-actin and the tight junction's protein ZO-1, whichaccompanies the increased paracellular permeability (Drug Deliv. Rev.2001; 50:S91-S111). It is suggested that an interaction between chitosanand the tight junction protein ZO-1, leads to its translocation to thecytoskeleton.

After removal of the incubated nanoparticles, a gradual increase in TEERvalues was noticed. This phenomenon indicated that the intercellulartight junctions of Caco-2 cell monolayers started to recover gradually;however, the TEER values did not recover to their initial values (FIG.6). Kotze et al. reported that complete removal of a CS-derived polymer,without damaging the cultured cells, was difficult due to the highlyadhesive feature of CS (Pharm. Res. 1997; 14:1197-1202). This might bethe reason why the TEER values did not recover to their initial values.In contrast, the TEER values of Caco-2 cell monolayers incubated withthe nanoparticles with a negative surface charge (γ-PGA dominated on thesurface, 0.10% γ-PGA:0.01% CS and 0.20% γ-PGA:0.01% CS, FIG. 6) showedno significant differences as compared to the control group (p>0.05).This indicated that γ-PGA does not have any effects on the opening ofthe intercellular tight junctions.

FIG. 8 shows an illustrative protein transport mechanism through acellular layer, including transcellular transport and paracellulertransport. FIG. 9 shows a schematic illustration of a paracellulartransport mechanism. The transcellular protein or peptide transport maybe an active transport or a passive transport mode whereas theparacellular transport is basically a passive mode. Ward et al. reportedand reviewed current knowledge regarding the physiological regulation oftight junctions and paracellular permeability (PSTT 2000; 3:346-358).Chitosan as nanoparticle vehicles for oral delivery of protein drugsavoids the enzymatic inactivation in the gastrointestinal conduit. Thechitosan component of the present nanoparticles has a special feature ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells; that is, loosening the tightness ofthe tight junctions.

FIG. 9(A) shows that after feeding nanoparticles (NPs) orally, NPsadhere and infiltrate into the mucus layer of the epithelial cells. FIG.9(B) illustrates that the infiltrated NPs transiently and reversiblyloosen tight junctions (TJs) while becoming unstable and disintegratedto release insulin or entrapped agent. FIG. 9( c) shows that thereleased insulin or agent permeates through the paracellular pathwayinto the blood stream. Chitosan (CS), a nontoxic, soft-tissuecompatible, cationic polysaccharide has special features of adhering tothe mucosal surface; CS is able to transiently and reversiblywiden/loosen TJs between epithelial cells. The TJ width in the smallintestine has been demonstrated to be less than 1 nm. It is also knownthat TJs ‘opened’ by absorption enhancers are less than 20 nm wide(Nanotechnology 2007; 18:1-11). The term “opened” herein means that anysubstance less than 20 nm in the close-proximity might have the chanceto pass through. TJs constitute the principal barrier to passivemovement of fluid, electrolytes, macromolecules and cells through theparacellular pathway.

It was suggested that the electrostatic interaction between thepositively charged CS and the negatively charged sites of ZO-1 proteinson cell surfaces at TJ induces a redistribution of cellular F-actin andZO-1's translocation to the cytoskeleton, leading to an increase inparacellular permeability. As evidenced in FIG. 9, after adhering andinfiltrating into the mucus layer of the duodenum, the orallyadministered nanoparticles may degrade due to the presence of distinctdigestive enzymes in the intestinal fluids. Additionally, the pHenvironment may become neutral while the nanoparticles were infiltratinginto the mucosa layer and approaching the intestinal epithelial cells.This further leads to the collapse of nanoparticles due to the change inthe exposed pH environment. The dissociated CS from thedegraded/collapsed nanoparticles was then able to interact and modulatethe function of ZO-1 proteins between epithelial cells (Nanotechnology2007; 18:1-11). ZO-1 proteins are thought to be a linkage moleculebetween occludin and F-actin cytoskeleton and play important roles inthe rearrangement of cell-cell contacts at TJs.

Example No. 7 fCS-γ-PGA Nanoparticle Preparation and CLSM Visualization

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles (FIG. 10)were prepared for the confocal laser scanning microscopy (CLSM) study.The nanoparticles of the present invention display a structure of aneutral polyelectrolyte-complex core surrounded by a positively chargedchitosan shell. Synthesis of the FITC-labeled low-MW CS (fCS) was basedon the reaction between the isothiocyanate group of FITC and the primaryamino groups of CS as reported in the literature (Pharm. Res. 2003;20:1812-1819). Briefly, 100 mg of FITC in 150 ml of dehydrated methanolwere added to 100 ml of 1% low-MW CS in 0.1M acetic acid. After 3 hoursof reaction in the dark at ambient conditions, fCS was precipitated byraising the pH to about 8-9 with 0.5M NaOH. To remove the unconjugatedFITC, the precipitate was subjected to repeated cycles of washing andcentrifugation (40,000×g for 10 min) until no fluorescence was detectedin the supernatant. The fCS dissolved in 80 ml of 0.1M acetic acid wasthen dialyzed for 3 days in the dark against 5 liters of distilledwater, with water replaced on a daily basis. The resultant fCS waslyophilized in a freeze dryer. The fCS-γ-PGA nanoparticles were preparedas per the procedure described in Example No. 4.

Subsequently, the transport medium containing fCS-γ-PGA nanoparticles(0.2 mg/ml) was introduced into the donor compartment of Caco-2 cells,which were pre-cultured on the transwell for 18-21 days. Theexperimental temperature was maintained at 37° C. by a temperaturecontrol system (DH-35 Culture Dish Heater, Warner Instruments Inc.,Hamden, Conn.). After incubation for specific time intervals, testsamples were aspirated. The cells were then washed twice with pre-warmedPBS solution before they were fixed in 3.7% paraformaldehyde (Pharm.Res. 2003; 20:1812-1819). Cells were examined under an inversed CLSM(TCS SL, Leica, Germany). The fluorescence images were observed using anargon laser (excitation at 488 nm, emission collected at a range of510-540 nm).

CLSM was used to visualize the transport of the fluorescence-labeledCS-γ-PGA (fCS-γ-PGA) nanoparticles across the Caco-2 cell monolayers.This non-invasive method allows for optical sectioning and imaging ofthe transport pathways across the Caco-2 cell monolayers, withoutdisrupting their structures (J. Control. Release 1996; 39:131-138).FIGS. 7 a and 7 b show the fluorescence images of 4 optical sections ofa Caco-2 cell monolayer that had been incubated with the fCS-γ-PGAnanoparticles having a positive surface charge (0.10% γ-PGA:0.20% CS,zeta potential: about 21 mV) for 20 and 60 min, respectively. As shown,after 20 min of incubation with the nanoparticles, intense fluorescencesignals at intercellular spaces were observed at depths of 0 and 5 μmfrom the apical (upper) surface of the cell monolayer. The intensity offluorescence became weaker at levels deeper than 10 μm from the apicalsurface of the cell monolayer and was almost absent at depths ≧15 μm(FIG. 7 a).

After 60 minutes of incubation with the nanoparticles, the intensity offluorescence observed at intercellular spaces was stronger and appearedat a deeper level than those observed at 20 min after incubation. Theseobservations confirmed with our TEER results that the nanoparticles witha positive surface charge (CS dominated on the surface) were able toopen the tight junctions between Caco-2 cells and allowed transport ofthe nanoparticles by passive diffusion via the paracellular pathways.

Example No. 8 In Vivo Study with Fluorescence-Labeled Nanoparticles

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles wereprepared for the confocal laser scanning microscopy (CLSM) study. Afterfeeding rats with fCS-γ-PGA nanoparticles, the rats are sacrificed at apre-determined time and the intestine is isolated for CLSM examination.The fluorescence images of the nanoparticles were clearly observed byCLSM that penetrates through the mouse intestine at appropriate time andat various depths from the inner surface toward the exterior surface ofthe intestine, including duodenum, jejunum, and ileum.

Example No. 9 Insulin Loading Capacity in Nanoparticles

Fluorescence (FITC)-labeled γ-PGA was added into chitosan solution toprepare fluorescence (FITC)-labeled, insulin-loaded CS-γ-PGAnanoparticles for in vivo animal study with confocal laser scanningmicroscopy (CLSM) assessment and bioactivity analysis. Theinsulin-loaded CS-γ PGA nanoparticles are by using the ionic-gelationmethod upon addition of insulin mixed with γ-PGA solution into CSsolution, followed by magnetic stirring in a container.

Model insulin used in the experiment and disclosed herein is obtainedfrom bovine pancreas (Sigma-Aldrich, St. Louis, Mo.), having a molecularformula of C₂₅₄H₃₇₇N₆₅O₇₅S₆ with a molecular weight of about 5733.5 andan activity of >27 USP units/mg. The insulin contains two-chainpolypeptide hormone produced by the β-cells of pancreatic islets. The αand β chains are joined by two interchain disulfide bonds. Insulinregulates the cellular uptake, utilization, and storage of glucose,amino acids, and fatty acids and inhibits the breakdown of glycogen,protein, and fat. The insulin from Sigma-Aldrich contains about 0.5%zinc. Separately, insulin can be obtained from other sources, such ashuman insulin solution that is chemically defined, recombinant fromSaccharomyces cerevisiae. Some aspects of the invention relate tonanoparticles with insulin in the core, wherein the insulin may containintermediate-acting, regular insulin, rapid-acting insulin,sustained-acting insulin that provides slower onset and longer durationof activity than regular insulin, or combinations thereof.

Examples of insulin or insulin analog products include, but not limitedto, Humulin® (by Eli Lilly), Humalog® (by Eli Lilly) and Lantus® (byAventis), and Novolog® Mix70/30 (by Novo Nordisk). Humalog (insulinlispro, rDNA origin) is a human insulin analog that is a rapid-acting,parenteral blood glucose-lowering agent. Chemically, it is Lys(B28),Pro(B29) human insulin analog, created when the amino acids at positions28 and 29 on the insulin B-chain are reversed. Humalog is synthesized ina special non-pathogenic laboratory strain of Escherichia coli bacteriathat has been genetically altered by the addition of the gene forinsulin lispro. Humalog has the empirical formula C₂₅₇H₃₈₃N₆₅O₇₇S₆ and amolecular weight of 5808, identical to that of human insulin. The vialsand cartridges contain a sterile solution of Humalog for use as aninjection. Humalog injection consists of zinc-insulin lispro crystalsdissolved in a clear aqueous fluid. Each milliliter of Humalog injectioncontains insulin lispro 100 Units, 16 mg glycerin, 1.88 mg dibasicsodium phosphate, 3.15 mg m-cresol, zinc oxide content adjusted toprovide 0.0197 mg zinc ion, trace amounts of phenol, and water forinjection. Insulin lispro has a pH of 7.0-7.8. Hydrochloric acid 10%and/or sodium hydroxide 10% may be added to adjust pH.

Humulin is used by more than 4 million people with diabetes around theworld every day. Despite its name, this insulin does not come from humanbeings. It is identical in chemical structure to human insulin and ismade in a factory using a chemical process called recombinant DNAtechnology. Humulin L is an amorphous and crystalline suspension ofhuman insulin with a slower onset and a longer duration of activity (upto 24 hours) than regular insulin. Humulin U is a crystalline suspensionof human insulin with zinc providing a slower onset and a longer andless intense duration of activity (up to 28 hours) than regular insulinor the intermediate-acting insulins (NPH and Lente).

LANTUS® (insulin glargine [rDNA origin] injection) is a sterile solutionof insulin glargine for use as an injection. Insulin glargine is arecombinant human insulin analog that is a long-acting (up to 24-hourduration of action), parenteral blood-glucose-lowering agent. LANTUS isproduced by recombinant DNA technology utilizing a non-pathogeniclaboratory strain of Escherichia coli (K12) as the production organism.Insulin glargine differs from human insulin in that the amino acidasparagine at position A21 is replaced by glycine and two arginines areadded to the C-terminus of the B-chain. Chemically, it is 2A-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has the empiricalformula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063.

LANTUS consists of insulin glargine dissolved in a clear aqueous fluid.Each milliliter of LANTUS (insulin glargine injection) contains 100 IU(3.6378 mg) insulin glargine. Inactive ingredients for the 10 mL vialare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, 20 mcg polysorbate20, and water for injection. Inactive ingredients for the 3 mL cartridgeare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, and water forinjection. In 2006, there were 11.4 million prescriptions of Lantus inthe U.S. for basal insulin maintenance.

Novolog® Mix70/30 (70% insulin aspart protamine suspension and 30%insulin aspart injection [rDNA origin]) is a human insulin analogsuspension. Novolog® Mix70/30 is a blood glucose-lowering agent with arapid onset and an intermediate duration of action. Insulin aspart ishomologous with regular human insulin with the exception of a singlesubstitution of the amino acid praline by aspartic acid in position B28,and is produced by recombinant DNA technology utilizing Saccharomycescerevisiae as the production organism. Insulin aspart (Novolog) has theempirical formula C₂₅₆H₃₈₁N₆₅O₇₉S₆ and a molecular weight of 5826.Novolog® Mix70/30 is a uniform, white sterile suspension that containszinc 19.6 μg/ml and other components.

The nanoparticles with two insulin concentrations are prepared at achitosan to γ-PGA ratio of 0.75 mg/ml to 0.167 mg/ml. Their particlesize and zeta potential are shown in Table 3 below.

TABLE 3 Insulin Conc. Mean Particle Polydispersity Zeta Potential(mg/ml) (n = 5) Size (nm) Index (PI) (mV) 0* 145.6 ± 1.9 0.14 ± 0.01+32.11 ± 1.61 0.042 185.1 ± 5.6 0.31 ± 0.05 +29.91 ± 1.02 0.083 198.4 ±6.2 0.30 ± 0.09 +27.83 ± 1.22 *control reference without insulin

Further, their association efficiency of insulin and loading capacity ofinsulin are analyzed, calculated and shown in FIGS. 11 and 12, accordingto the following formula:

${{Insulin}\mspace{14mu} {Association}\mspace{14mu} {{Efficiency}\left( {L\; E\mspace{14mu} \%} \right)}} = {\frac{\begin{pmatrix}{{{Total}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {insulin}} -} \\{{Insulin}\mspace{14mu} {in}\mspace{14mu} {supernatant}}\end{pmatrix}}{{Total}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {insulin}} \times 100\%}$${{Loading}\mspace{14mu} {{Capacity}\left( {L\; C} \right)}} = {\frac{\begin{pmatrix}{{{Total}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {insulin}} -} \\{{Insulin}\mspace{14mu} {in}\mspace{14mu} {supernatant}}\end{pmatrix}}{{Weight}\mspace{14mu} {of}\mspace{14mu} {recovered}\mspace{14mu} {particles}} \times 100\%}$

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA, whereas FIG. 12 shows loadingcapacity and association efficiency of insulin in nanoparticles ofchitosan alone (in absence of γ-PGA) as reference. The data clearlydemonstrates that both the insulin loading capacity and insulinassociation efficiency are statistically higher for the nanoparticleswith γ-PGA in the core. The LE (40˜55%) and LC (5.0˜14.0%) of insulinfor CS-γPGA nanoparticles was obtained by using ionic-gelation methodupon addition of insulin mixed with γ-PGA solution into CS solution,followed by magnetic stirring for nanoparticle separation.

In certain follow-up experiments, nanoparticles having a pharmaceuticalcomposition have been successfully prepared with a negatively chargedcomponent comprised of γ-PGA, α-PGA, PGA derivatives, salts of PGA,heparin or heparin analog, glycosaminoglycans, or alginate. The PGAderivatives of the present invention may include, but not limited to,poly-γ-glutamic acid, poly-α-glutamic acid, poly-L-glutamic acid(manufactured by Sigma-Aldrich, St. Louis, Mo.), poly-D-glutamic acid,poly-L-α-glutamic acid, poly-γ-D-glutamic acid, poly-γ-DL-glutamic acid,and PEG or PHEG derivatives of polyglutamic acid, salts of theabove-cited PGAs, and the like. Some aspects of the invention relate tonanoparticles comprising a shell component and a core component, whereinat least a portion of the shell component comprises chitosan and whereinthe core component is comprised of a negatively charged compound that isconjugated to chitosan, and a bioactive agent. Some aspects of theinvention relate to an oral dose of nanoparticles that effectivelyenhance intestinal or blood brain paracellular transport comprising anegative component (such as γ-PGA, α-PGA, PGA derivatives, heparin, oralginate) in the core and low molecular weight chitosan, wherein thechitosan dominates on a surface of the nanoparticles with positivecharges.

Some aspects of the invention relate to a dose of nanoparticles thateffectively enhance intestinal or blood brain paracellular transportcomprising a polyanionic component (such as γ-PGA, α-PGA, PGAderivatives, heparin, heparin analogs, low molecular weight heparin,glycosaminoglycans, or alginate) in the core and low molecular weightchitosan in the shell, wherein the chitosan dominates on a surface ofthe nanoparticles with surface positive charges. In use, firstly,encapsulate the Alzheimer's drug in the chitosan shell nanoparticle asdescribed herein, wherein the nanoparticle is partially crosslinked(optionally) to enhance its biodurability. Then intra-venously injectthe nanoparticles, whereby the nanoparticles pass to the brain in bloodcirculation. The chitosan shell of the nanoparticles adheres to thesurface adjacent the tight junction in the brain. Thereafter, thechitosan nanoparticle opens the tight junction, wherein the Alzheimer'sdrug is released after passing the tight junction for therapeutictreatment. In one embodiment, the nanoparticles are in a spherical shapehaving a mean particle size of about 50 to 250 nanometers, preferably150 nanometers to 250 nanometers.

In one example, intravenous administration of the nanoparticlescomprising chitosan shell substrate, polyanionic core substrate and atleast one bioactive agent for treating Alzheimer's disease in a patientis typically performed with 10 mg to 40 mg of active agent per day overa period of one month to one year. The bioactive agent is selected fromthe group consisting of donepezile, rivastignine, galantamine, and/orthose trade-named products, such as memantine hydrochloride (Axura® byMerz Pharmaceuticals), donepezil hydrochloride (Aricept® by Eisai Co.Ltd.), rivastigmine tartrate (Exelon® by Novartis), galantaminehydrochloride (Reminyl® by Johnson & Johnson), and tacrine hydrochloride(Cognex® by Parke Davis).

Some aspects of the invention relate to a nanoparticle with a coresubstrate comprising polyglutamic acids such as water soluble salt ofpolyglutamic acids (for example, ammonium salt) or metal salts ofpolyglutamic acid (for example, lithium salt, sodium salt, potassiumsalt, magnesium salt, and the like). In one embodiment, the form ofpolyglutamic acid may be selected from the group consisting ofpoly-α-glutamic acid, poly-L-α-glutamic acid, poly-γ-glutamic acid,poly-D-glutamic acid, poly-γ-D-glutamic acid, poly-γ-DL-glutamic acid,poly-L-glutamic acid (manufactured by Sigma-Aldrich, St. Louis, Mo.),and PEG or PHEG derivatives of polyglutamic acid. Alginate is generallynon-biodegradable; however, it is stipulated that an alginate particlewith about 30-50 kDa molecular weight is kidney inert. Heparin withnegatively charged side-groups has a general chemical structure as shownbelow:

Some aspects of the invention relate to the negatively chargedglycosaminoglycans (GAGs) as the core substrate of the presentnanoparticles. GAGs may be used to complex with a low-molecular-weightchitosan to form drug-carrier nanoparticles. GAGs may also conjugatewith the protein drugs as disclosed herein to enhance the bondingefficiency of the core substrate in the nanoparticles. Particularly, thenegatively charged core substrate (such as GAGs, heparin, PGA, alginate,and the like) of the nanoparticles of the present invention mayconjugate with chondroitin sulfate, hyaluronic acid, PDGF-BB, BSA, EGF,MK, VEGF, KGF, bFGF, aFGF, MK, PTN, etc.

Calceti et al. reported an in vivo evaluation of an oral insulin-PEGdelivery system (Eur J Pharma Sci 2004; 22:315-323). Insulin-PEG wasformulated into mucoadhesive tablets constituted by the thiolatedpolymer poly(acrylic acid)-cysteine. The therapeutic agent was sustainedreleased from these tablets within 5 hours. In vivo, by oraladministration to diabetic mice, the glucose levels were found todecrease significantly over the time. Further, Krauland et al. reportedanother oral insulin delivery study of thiolated chitosan-insulintablets on non-diabetic rats (J. Control. Release 2004, 95:547-555). Thedelivery tablets utilized 2-Iminothiolane covalently linked to chitosanto form chitosan-TBA (chitosan-4-thiobutylamidine) conjugate. After oraladministration of chitosan-TBA-insulin tablets to non-diabetic consciousrats, the blood glucose level decreased significantly for 24 hours;supporting the observation of sustained insulin release of the presentlydisclosed nanoparticles herein through intestinal absorption. In afurther report by Morcol et al. (Int. J. Pharm. 2004; 277:91-97), anoral delivery system comprising calcium phosphate-PEG-insulin-caseinparticles displays a prolonged hypoglycemic effect after oraladministration to diabetic rats.

Pan et al. disclosed chitosan nanoparticles improving the intestinalabsorption of insulin in vivo (Int J Pharma 2002; 249:139-147) withinsulin-chitosan nanoparticles at a particle size of 250-400 nm, apolydispersity index smaller than 0.1, positively charged and stable.After administering the insulin-chitosan nanoparticles, it was foundthat the hypoglycemic was prolonged with enhanced pharmacologicalbioavailability. Their data confirmed our observation as shown in FIGS.11 and 12; however, the insulin loading capacity and insulin associationefficiency of the present invention are substantially higher for thechitosan-insulin nanoparticles with γ-PGA in the core as the coresubstrate.

Example No. 10 Insulin Nanoparticle Stability

FIG. 13 shows the stability of insulin-loaded nanoparticles of thepresent invention with an exemplary composition of CS 0.75 mg/ml, γ-PGA0.167 mg/ml, and insulin 0.083 mg/ml. The prepared insulin-loadednanoparticles suspended in deionized water are stable during storage upto 40 days. First (in FIG. 13), the insulin content in the nanoparticlestorage solution maintains at about a constant level of 9.5%. Thenanoparticle stability is further evidenced by the substantiallyconstant particle size at about 200 nm and substantially constant zetapotential of about +28 mV over the period of about 40 days. It iscontemplated that the insulin-containing nanoparticles of the presentinvention would further maintain their biostability when formulated in asoft gelcap or capsule configuration that further isolates thenanoparticles from environmental effects, such as sunlight, heat, airconditions, and the like. Some aspects of the invention provide a gelcappill or capsule containing a dosage of insulin nanoparticles effectiveamount of the insulin to treat or manage the diabetic of an animalsubject, including a patient, wherein the stability of theinsulin-containing nanoparticles is at least 40 days, preferably morethan 6 months, and most preferably more than a couple of years.

By “effective amount of the insulin”, it is meant that a sufficientamount of insulin will be present in the dose to provide for a desiredtherapeutic, prophylatic, or other biological effect when thecompositions are administered to a host in the single dosage forms. Thecapsule of the present invention may preferably comprise two-parttelescoping gelatin capsules. Basically, the capsules are made in twoparts by dipping metal rods in molten gelatin solution. The capsules aresupplied as closed units to the pharmaceutical manufacturer. Before use,the two halves are separated, the capsule is filled with powder (eitherby placing a compressed slug of powder into one half of the capsule, orby filling one half of the capsule with loose powder) and the other halfof the capsule is pressed on. The advantage of inserting a slug ofcompressed powder is that control of weight variation is better. Thecapsules may be enterically coated before filling the powder or afterfilling the powder and securing both parts together.

Thus, for convenient and effective oral administration, pharmaceuticallyeffective amounts of the nanoparticles of this invention can betabletted with one or more excipient, encased in capsules such as gelcapsules, and suspended in a liquid solution and the like. Thenanoparticles can be suspended in a deionized solution or the like forparenteral administration. The nanoparticles may be formed into a packedmass for ingestion by conventional techniques. For instance, thenanoparticles may be encapsulated as a “hard-filled capsule” or a“soft-elastic capsule” using known encapsulating procedures andmaterials. The encapsulating material should be highly soluble ingastric fluid so that the particles are rapidly dispersed in the stomachafter the capsule is ingested. Each unit dose, whether capsule ortablet, will preferably contain nanoparticles of a suitable size andquantity that provides pharmaceutically effective amounts of thenanoparticles. The applicable shapes and sizes of capsules may includeround, oval, oblong, tube or suppository shape with sizes from 0.75 mmto 80 mm or larger. The volume of the capsules can be from 0.05 cc tomore than 5 cc. In one embodiment, the interior of capsules is treatedto be hydrophobic or lipophilic.

Example No. 11 In Vitro Insulin Release Study

FIG. 14 show a representative protein drug (for example, insulin)release profile in a pH-adjusted solution for pH-sensitivity study withan exemplary composition of CS 0.75 mg/ml, γ-PGA 0.167 mg/ml, andinsulin 0.083 mg/ml in nanoparticles. In one embodiment, the exemplarycomposition may include each component at a concentration range of +10%as follows: CS 0.75 mg/ml (a concentration range of 0.67 to 0.83 mg/ml),γ-PGA 0.167 mg/ml (a concentration range of 0.150 to 0.184 mg/ml), andinsulin 0.083 mg/ml (a concentration range of 0.075 to 0.091 mg/ml).First, solution of the insulin-loaded nanoparticles was adjusted to pH2.5 to simulate the gastric environment in a DISTEK-2230A container at37° C. and 100 rpm. Samples (n=5) were taken at a pre-determinedparticular time interval and the particle-free solution was obtained bycentrifuging at 22,000 rpm for 30 minutes to analyze the free orreleased insulin in solution by HPLC. Until the free insulin content inthe sample solution approaches about constant of 26% (shown in FIG. 14),the pH was adjusted to 6.6 to simulate the entrance portion of theintestine. The net released insulin during this particular time intervalis about (from 26% to 33%) 7%. In other words, the nanoparticles arequite stable (evidenced by minimal measurable insulin in solution) forboth the pH 2.5 and pH 6.6 regions. To further simulate the exit portionof the intestine, the insulin-containing nanoparticle solution isadjusted to pH 7.4. The remaining insulin (about 67%) is released fromthe nanoparticles. As discussed above, the insulin in nanoparticleswould be more effective to penetrate the intestine wall in paracellulartransport mode than the free insulin because of the nanoparticles of thepresent invention with chitosan at the outer surface (preferentialmucosal adhesion on the intestinal wall) and positive charge (enhancingparacellular tight junction transport).

Example No. 12 In Vivo Study with Insulin-Loaded Fluorescence-LabeledNanoparticles

In the in vivo study, rats were injected with streptozotocin (STZ 75mg/kg intraperitoneal) in 0.01M citrate buffer (pH 4.3) to inducediabetes rats. The blood from the rat's tail was analyzed with acommercially available glucometer for blood glucose. The blood glucoselevel on Wistar male rats at no fasting (n=5) is measured at 107.2±8.1mg/dL for normal rats while the blood glucose level is at 469.7±34.2mg/dL for diabetic rats. In the animal study, diabetic rats were fastingfor 12 hours and subjected to four different conditions: (a) oraldeionized water (DI) administration; (b) oral insulin administration at30 U/kg; (c) oral insulin-loaded nanoparticles administration at 30U/kg; and (d) subcutaneous (SC) insulin injection at 5 U/kg as positivecontrol. The blood glucose concentration from rat's tail was measuredover the time in the study.

FIG. 15 shows glucose change (hypoglycemic index) versus time of the invivo animal study (n=5). The glucose change as a percentage of baselines for both oral DI administration and oral insulin administrationover a time interval of 8 hours appears relatively constant within theexperimental measurement error range. It is illustrative thatsubstantially all insulin from the oral administration route has beendecomposed in rat stomach. As anticipated, the glucose decrease for theSC insulin injection route appears in rat blood in the very early timeinterval and starts to taper off after 3 hours in this exemplary study.The most important observation of the study comes from the oraladministration route with insulin-loaded nanoparticles.

The blood glucose begins to decrease from the base line at about 2 hoursafter administration and sustains at a lower glucose level at more than8 hours into study. It implies that the current insulin-loadednanoparticles may modulate the glucose level in animals in a sustainedor prolonged effective mode. Some aspects of the invention provide amethod of treating diabetes of an animal subject, including a patient,comprising orally administering insulin-containing nanoparticles with adosage effective amount of the insulin to treat the diabetes, wherein atleast a portion of the nanoparticles comprises a positively chargedshell substrate and a negatively charged core substrate. In oneembodiment, the dosage effective amount of the insulin to treat thediabetes comprises an insulin amount of between about 15 units to 45units per kilogram body weight of the patient or animal subject,preferably 20 to 40 units, and most preferably at about 25 to 35 unitsinsulin per kilogram body weight.

Some aspects of the invention relate to a novel nanoparticle system thatis composed of a low-MW CS and γ-PGA with CS dominated on the surfacesbeing configured to effectively open the tight junctions between Caco-2cell monolayers. The surface of the nanoparticles is characterized witha positive surface charge. In one embodiment, the nanoparticles of theinvention enables effective intestinal delivery for bioactive agent,including peptide, polypeptide, protein drugs, other large hydrophilicmolecules, and the like. Such polypeptide drugs can be any natural orsynthetic polypeptide that may be orally administered to a humanpatient.

Exemplary drugs include, but are not limited to, insulin; growthfactors, such as epidermal growth factor (EGF), insulin-like growthfactor (IGF), transforming growth factor (TGF), nerve growth factor(NGF), platelet-derived growth factor (PDGF), bone morphogenic protein(BMP), fibroblast growth factor and the like; somatostatin;somatotropin; somatropin; somatrem; calcitonin; parathyroid hormone;colony stimulating factors (CSF); clotting factors; tumor necrosisfactors: interferons; interleukins; gastrointestinal peptides, such asvasoactive intestinal peptide (VIP), cholecytokinin (CCK), gastrin,secretin, and the like; erythropoietins; growth hormone and GRF;vasopressins; octreotide; pancreatic enzymes; dismutases such assuperoxide dismutase; thyrotropin releasing hormone (TRH); thyroidstimulating hormone; luteinizing hormone; LHRH; GHRH; tissue plasminogenactivators; macrophage activator; chorionic gonadotropin; heparin;atrial natriuretic peptide; hemoglobin; retroviral vectors; relaxin;cyclosporin; oxytocin; vaccines; monoclonal antibodies; and the like;and analogs and derivatives of these compounds.

The bioactive agent of the present invention may also be selected fromgroup consisting of oxytocin, vasopressin, adrenocorticotrophic hormone,prolactin, luliberin or luteinising hormone releasing hormone, growthhormone, growth hormone releasing factor, somatostatin, glucagon,interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin,calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin,bacitracins, polymixins, colistins, tyrocidin, gramicidines, andsynthetic analogues, modifications and pharmacologically activefragments thereof, monoclonal antibodies and soluble vaccines.

In another embodiment, the nanoparticles of the invention increase theabsorption of bioactive agents across the blood brain barrier and/or thegastrointestinal barrier. In still another embodiment, the nanoparticleswith chitosan at an outer layer and surface positive charge serve as anenhancer in enhancing paracellular drug (bioactive agent) transport ofan administered bioactive agent when the bioactive agent andnanoparticles are orally administrated in a two-component system, ororally administered substantially simultaneously.

Example No. 13 Paracellular Transport and Enhancers

Chitosan and its derivatives may function as intestinal absorptionenhancers (that is, paracellular transport enhancers). Chitosan, whenprotonated at an acidic pH, is able to increase the paracellularpermeability of peptide drugs across mucosal epithelia. Some aspects ofthe invention provide co-administration of nanoparticles of the presentinvention and at least one paracellular transport enhancer (innon-nanoparticle form or nanoparticle form). In one embodiment, thenanoparticles can be formulated by co-encapsulation of the at least oneparacellular transport enhancer and at least one bioactive agent,optionally with other components. The absorption enhancer may beselected from the group consisting of Ca²⁺ chelators, bile salts,anionic surfactants, medium-chain fatty acids, phosphate esters, andchitosan or chitosan derivatives. In one embodiment, the nanoparticlesof the present invention comprises a positively charged shell substrateand a negatively charged core substrate, for example, nanoparticlescomposed of γ-PGA and chitosan that is characterized with asubstantially positive surface charge.

In some embodiment, the nanoparticles of the present invention and theat least one paracellular transport enhancer are encapsulated in a softgel, pill, or enteric coated capsule. The enhancers and thenanoparticles would arrive at the tight junction about the same time forenhancing opening the tight junction. In another embodiment, the atleast one paracellular transport enhancer is co-enclosed within theshell of the nanoparticles of the present invention. Therefore, somebroken nanoparticles would release enhancers to assist the nanoparticlesto open the tight junctions of the epithelial layers. In an alternateembodiment, the at least one enhancer is enclosed within a secondnanoparticle having positive surface charges, particularly a chitosantype nanoparticle. When the drug-containing first nanoparticles of thepresent invention are co-administered with the above-identified secondnanoparticles orally, the enhancers within the second nanoparticles arereleased in the intestinal tract to assist the drug-containing firstnanoparticles to open and pass the tight junction.

Example No. 14 Nanoparticles with Exenatide

Exenatide is a member of the class of drugs known as incretin mimetics.Exenatide and pramlintide belong to non-insulin injectables fortreatment of diabetes. Exenatide has a molecular formula ofC₁₈₄H₂₈₂N₅₀O₆₀S with a molecular mass of about 4186.6 g/mol and an CASno. 141732-76-5. Exenatide is suitable to be incorporated in a coreportion of chitosan-shelled nanoparticles, wherein the core portion mayinclude positively charged chitosan and negatively charged coresubstrate, such as γ-PGA or α-PGA, optionally with additional TPP andMgSO₄ in the core portion. In preparation, nanoparticles were obtainedupon addition of a mixture of γ-PGA plus exenatide aqueous solution (pH7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTechScientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10ml) at concentrations higher than 0.10% by w/v under magnetic stirringat room temperature to ensure positive surface charge. Nanoparticleswere collected by ultracentrifugation at 38,000 rpm for 1 hour.Exenatide is wholly or substantially totally encapsulated in the coreportion of the nanoparticles. Supernatants were discarded andnanoparticles were resuspended in deionized water as the solutionproducts. In one embodiment, it may further be encapsulated in capsules.In one embodiment, the interior surface of the capsule is treated to belipophilic or hydrophobic. In another embodiment, the exterior surfaceof the capsule is enteric-coated. In a preferred embodiment, thenanoparticles are further freeze-dried, optionally being mixed withtrehalose or with hexan-1,2,3,4,5,6-hexyl in a freeze-drying process.

Glucagon-like peptide-1 (GLP-1) is derived from the transcriptionproduct of the proglucagon gene. The major source of GLP-1 in the bodyis the intestinal L cell that secretes GLP-1 as a gut hormone. Thebiologically active forms of GLP-1 are GLP-1-(7-37) and GLP-1-(7-36)NH2.GLP-1 secretion by L cells is dependent on the presence of nutrients inthe lumen of the small intestine. The secretagogues (agents that causeor stimulate secretion) of this hormone include major nutrients likecarbohydrate, protein and lipid. Once in the circulation, GLP-1 has ahalf-life of less than 2 minutes, due to rapid degradation by the enzymedipeptidyl peptidase-4 (DPP-4). Commercial GLP-1 ELISA kits aregenerally available for GLP-1 assay.

Exenatide (marketed as Byetta) is the first of a new class ofmedications (incretin mimetics) approved for the treatment of type 2diabetes. It is manufactured and marketed by Amylin Pharmaceuticals andEli Lilly and Company. Exenatide is a synthetic version of exendin-4, ahormone in the saliva of the Gila monster, a lizard native to severalSouthwestern American states. It displays properties similar to humanGLP-1. Exenatide is a 39-amino-acid peptide that mimics the GLP-1incretin, an insulin secretagogue with glucoregulatory effects. While itmay lower blood glucose levels on its own, it can also be combined withother medications such as pioglitazone, metformin, sulfonylureas, and/orinsulin (not FDA approved yet) to improve glucose control. The approveduse of exenatide is with either sulfonylureas, metformin orthiazolinediones. The medication is injected subcutaneously twice perday using a pre-filled pen device.

Typical human responses to exenatide include improvements in the initialrapid release of endogenous insulin, suppression of pancreatic glucagonrelease, delayed gastric emptying, and reduced appetite—all of whichfunction to lower blood glucose. Whereas some other classes of diabetesdrugs such as sulfonylureas, thiazolinediones, and insulin are oftenassociated with weight gain, Byetta often is associated with significantweight loss. Unlike sulfonylureas and meglitinides, exenatide increasesinsulin synthesis and secretion in the presence of glucose only,lessening the risk of hypoglycemia. Byetta is also being used by somephysicians to treat insulin resistance. Some aspects of the inventionrelate to a pharmaceutical composition of nanoparticles for oraladministration in an animal subject, the nanoparticles comprising ashell portion that is dominated by positively charged chitosan, a coreportion that contains negatively charged substrate, wherein thenegatively charged substrate is at least partially neutralized with aportion of the positively charged chitosan in the core portion, and atleast one bioactive agent loaded within the nanoparticles, wherein thebioactive agent is exenatide or pramlintide. In one embodiment, thebioactive agent is a combination of exenatide with insulin orpramlintide with insulin.

Example No. 15 Nanoparticles with Pramlintide

Pramlintide is a synthetic amylin analogue (marketed as Symlin). Amylinis a natural, pancreatic islet peptide that is normally secreted withinsulin in response to meals. It has several beneficial effects onglucose homeostasis: suppression of glucagon secretion, delaying ofgastric emptying, and the promotion of satiety. It is currently givenbefore meals, in a separate subcutaneous injection but usually inconjunction with insulin. Pramlintide has a molecular formula ofC₁₇₁H₂₆₉N₅₁O₅₃S₂ with a molecular mass of about 3951.4 g/mol and an CASno. 151126-32-8. Pramlintide (positively charged) is currently deliveredas an acetate salt. Pramlintide is suitable to be incorporated in a coreportion of a chitosan-shelled nanoparticles, wherein the core portionmay include positively charged chitosan and negatively charged coresubstrate, such as γ-PGA or α-PGA, optionally with additional TPP andMgSO₄ in the core portion. In other words, pramlintide may replace atleast a portion of positively charged chitosan in the core portion byinteracting with negatively core substrate, such as PGA, heparin or thelike. In preparation, nanoparticles were obtained upon addition of amixture of γ-PGA plus ppramlintide aqueous solution (pH 7.4, 2 ml),using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc.,Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) atconcentrations higher than 0.10% by w/v under magnetic stirring at roomtemperature to ensure positive surface charge. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Pramlintideis wholly or substantially totally encapsulated in the core portion ofthe nanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water as the solution products. In oneembodiment, it may further be encapsulated in capsules. In oneembodiment, the interior surface of the capsule is treated to belipophilic or hydrophobic. In another embodiment, the exterior surfaceof the capsule is enteric-coated. In a preferred embodiment, thenanoparticles are further freeze-dried, optionally being mixed withtrehalose or with hexan-1,2,3,4,5,6-hexyl in a freeze-drying process.

Pramlintide is an analogue of amylin, a small peptide hormone that isreleased into the bloodstream by the β-cells of the pancreas along withinsulin, after a meal. Like insulin, amylin is deficient in individualswith diabetes. By augmenting endogenous amylin, pramlintide aids in theabsorption of glucose by slowing gastric emptying, promoting satiety viahypothalamic receptors (different receptors than for GLP-1), andinhibiting inappropriate secretion of glucagon, a catabolic hormone thatopposes the effects of insulin and amylin.

Example No. 16 Nanoparticles with Complexed Calcitonin

Calcitonin is a protein drug that serves therapeutically as calciumregulators for treating osteoporosis (J. Pharm. Pharmacol. 1994;46:547-552). Calcitonin has a molecular formula of C₁₄₅H₂₄₀N₄₄O₄₈S₂ witha molecular weight of about 3431.9 and an isoelectric point of 8.7. Thenet charge for calcitonin at pH7.4 is positive that is suitable tocomplex or conjugate with negatively charged core substrate, such asγ-PGA or α-PGA. In preparation, nanoparticles were obtained uponaddition of a mixture of γ-PGA plus calcitonin aqueous solution (pH 7.4,2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTech ScientificInc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) atconcentrations higher than 0.10% by w/v under magnetic stirring at roomtemperature to ensure positive surface charge. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Calcitonin iswholly or substantially totally encapsulated in the core portion of thenanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water as the solution products, furtherencapsulated in capsules or further treated with an enteric coating.

Example No. 17 Nanoparticles with Conjugated Vancomycin

Vancomycin is a protein drug that serves therapeutically as antibioticagainst bacterial pathogens. Vancomycin has a molecular formula ofC₆₆H₇₅N₉O₂₄ with a molecular weight of about 1485.7 and an isoelectricpoint of 5.0. The net charge for vancomycin at pH7.4 is negative that issuitable to complex or conjugate with a portion of negatively chargedshell substrate, such as chitosan. In preparation, nanoparticles wereobtained upon addition of a mixture of γ-PGA plus vancomycin aqueoussolution (pH 7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a low-MW CS aqueous solution(pH 6.0, 10 ml) with excess concentrations under magnetic stirring atroom temperature, wherein CS concentration is provided sufficiently toconjugate vancomycin, to counterbalance γ-PGA, and exhibit positivesurface charge for the nanoparticles. Nanoparticles were collected byultracentrifugation at 38,000 rpm for 1 hour. Vancomycin is wholly orsubstantially totally encapsulated in the core portion of thenanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water as the solution products, furtherencapsulated in capsules or further treated with an enteric coating oncapsules.

Some aspects of the invention relate to a method of enhancing intestinalor blood brain paracellular transport of bioactive agents configured andadapted for delivering at least one bioactive agent in an animalsubject, including a patient, comprising administering nanoparticlescomposed of γ-PGA and chitosan, wherein the nanoparticles are loadedwith a therapeutically effective amount or dose of the at least onebioactive agent. The nanoparticle of the present invention is aneffective intestinal delivery system for peptide and protein drugs andother large hydrophilic molecules. In a further embodiment, thebioactive agent is selected from the group consisting of proteins,peptides, nucleosides, nucleotides, antiviral agents, antineoplasticagents, antibiotics, and anti-inflammatory drugs. In a furtherembodiment, the bioactive agent is selected from the group consisting ofcalcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin,tyrotropin releasing hormone (TRH), follicle stimulating hormone (FSH),luteinizing hormone (LH), vasopressin and vasopressin analogs, catalase,superoxide dismutase, interleukin-II (IL2), interferon, colonystimulating factor (CSF), tumor necrosis factor (TNF) andmelanocyte-stimulating hormone. In a further embodiment, the bioactiveagent is an Alzheimer antagonist.

Example No. 18 Nanoparticles with Heparin Core Substrate

Heparin is a negatively charged drug that serves therapeutically asanti-coagulant. Heparin is generally administered by intravenousinjection. Some aspects of the invention relate to heparin nanoparticlesfor oral administration or subcutaneous administration. In a furtherembodiment, heparin serves as at least a portion of the core substratewith chitosan as shell substrate, wherein heparin conjugate at least onebioactive agent as disclosed herein. In preparation, nanoparticles wereobtained upon addition of heparin Leo aqueous solution (2 ml), using apipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany),into a low-MW CS aqueous solution (pH 6.0, 10 ml) with excessconcentrations under magnetic stirring at room temperature.Nanoparticles were collected by ultracentrifugation at 38,000 rpm for 1hour. Heparin is wholly or substantially totally encapsulated in thecore portion of the nanoparticles. Table 4 shows the conditions ofsolution preparation and the average nanoparticle size.

TABLE 4 Heparin Chitosan Particle Conditions conc. @2 ml conc. @10 mlsize (nm) A 200 iu/ml 0.09% 298.2 ± 9.3 B 100 iu/ml 0.09% 229.1 ± 4.5 C 50 iu/ml 0.09% 168.6 ± 1.7 D  25 iu/ml 0.09% 140.1 ± 2.3

To evaluate the pH stability of the heparin-containing nanoparticlesfrom Example no. 18, the nanoparticles from Condition D in Table 4 aresubjected to various pH for 2 hours (sample size=7). Table 5 shows theaverage size, size distribution (polydispersity index: PI) and zetapotential (Zeta) of the nanoparticles at the end of 2 hours undervarious pH environments. The data shows the nanoparticles are relativelystable. In one embodiment, the nanoparticles of the present inventionmay include heparin, heparin sulfate, small molecular weight heparin,and heparin derivatives.

TABLE 5 pH 1.5 2.6 6.6 7.4 Deionized water @5.9 Size (nm) 150 ± 9  160 ±12  153 ± 2  154 ± 4  147 ± 5  PI 0.54 ± 0.03 0.50 ± 0.04 0.08 ± 0.020.32 ± 0.03 0.37 ± 0.02 Zeta (+) 15 ± 2  33 ± 6   15 ± 0.1  11 ± 0.2 18± 4 

In a further embodiment, a growth factor such as bFGF withpharmaceutically effective amount is added to heparin Leo aqueoussolution before the pipetting step in Example No. 17. In our laboratory,growth factors and proteins with pharmaceutically effective amount havebeen successfully conjugated with heparin to form nanoparticles of thepresent invention with chitosan as the shell substrate, wherein thegrowth factor is selected from the group consisting of VascularEndothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor 2(VEGF2), basic Fibroblast Growth Factor (bFGF), Vascular EndothelialGrowth Factor 121 (VEGF121), Vascular Endothelial Growth Factor 165(VEGF165), Vascular Endothelial Growth Factor 189 (VEGF189), VascularEndothelial Growth Factor 206 (VEGF206), Platelet Derived Growth Factor(PDGF), Platelet Derived Angiogenesis Factor (PDAF), Transforming GrowthFactor-β (TGF-β), Transforming Growth Factor-α (TGF-α), Platelet DerivedEpidermal Growth Factor (PDEGF), Platelet Derived Wound Healing Formula(PDWHF), epidermal growth factor, insulin-like growth factor, acidicFibroblast Growth Factor (aFGF), human growth factor, and combinationsthereof; and the protein is selected from the group consisting ofhaemagglutinin (HBHA), Pleiotrophin, buffalo seminal plasma proteins,and combinations thereof.

In a co-pending application, U.S. patent application Ser. No. 10/916,170filed Aug. 11, 2004, it is disclosed that a biomaterial with free aminogroups of lysine, hydroxylysine, or arginine residues within biologictissues is crosslinkable with genipin, a crosslinker (Biomaterials 1999;20:1759-72). It is also disclosed that the crosslinkable biomaterial maybe crosslinked with a crosslinking agent or with light, such asultraviolet irradiation, wherein the crosslinkable biomaterial may beselected from the group consisting of collagen, gelatin, elastin,chitosan, NOCC (N, O, carboxylmethyl chitosan), fibrin glue, biologicalsealant, and the like. Further, it is disclosed that a crosslinkingagent may be selected from the group consisting of genipin, itsderivatives, analog (for example, aglycon geniposidic acid),stereoisomers and mixtures thereof. In one embodiment, the crosslinkingagent may further be selected from the group consisting of epoxycompounds, dialdehyde starch, glutaraldehyde, formaldehyde, dimethylsuberimidate, carbodiimides, succinimidyls, diisocyanates, acyl azide,reuterin, ultraviolet irradiation, dehydrothermal treatment,tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine andphoto-oxidizers, and the like.

In one embodiment, it is disclosed that loading drug onto achitosan-containing biological material crosslinked with genipin orother crosslinking agent may be used as biocompatible drug carriers fordrug slow-release or sustained release. Several biocompatible plasticpolymers or synthetic polymers have one or more amine group in theirchemical structures, for example poly(amides) or poly(ester amides). Theamine group may become reactive toward a crosslinking agent, such asglutaraldehyde, genipin or epoxy compounds of the present invention. Inone embodiment, the nanoparticles comprised of crosslinkable biomaterialis crosslinked, for example up to about 50% degree or more ofcrosslinking, preferably about 1 to about 20% degree of crosslinking ofthe crosslinkable components of the biomaterial, enabling sustainedbiodegradation of the biomaterial and/or sustained drug release.

By modifying the chitosan structure to alter its charge characteristics,such as grafting the chitosan with methyl, N-trimethyl, alkyl (forexample, ethyl, propyl, butyl, isobutyl, etc.), polyethylene glycol(PEG), or heparin (including low molecular weight heparin, regularmolecular weight heparin, and genetically modified heparin), the surfacecharge density (zeta potential) of the CS-γ PGA nanoparticles may becomemore pH resistant or hydrophilic. In one embodiment, the chitosan isgrafted with polyacrylic acid.

By way of illustration, trimethyl chitosan chloride might be used informulating the CS-γ PGA nanoparticles for maintaining its sphericalbiostability at a pH lower than pH 2.5, preferably at a pH as low as1.0. Some aspects of the invention provide a drug-loadedchitosan-containing biological material crosslinked with genipin orother crosslinking agent as a biocompatible drug carrier for enhancingbiostability at a pH lower than pH 2.5, preferably within at a pH as lowas 1.0.

Freeze-Dried Nanoparticles

A pharmaceutical composition of nanoparticles of the present inventionmay comprise a first component of at least one bioactive agent, a secondcomponent of chitosan (including regular molecular weight and lowmolecular weight chitosan), and a third component that is negativelycharged. In one embodiment, the second component dominates on a surfaceof the nanoparticle. In another embodiment, the chitosan is N-trimethylchitosan. In still another embodiment, the low molecular weight chitosanhas a molecular weight lower than that of a regular molecular weightchitosan. The nanoparticles may further comprise tripolyphosphate andmagnesium sulfate. For example, a first solution of (2 ml 0.1% γ-PGAaqueous solution @pH 7.4+0.05% Insulin +0.1% Tripolyphosphate (TPP)+0.2%MgSO₄) is added to a base solution (10 ml 0.12% chitosan aqueoussolution @pH 6.0) as illustrated in Example no. 4 under magneticstirring at room temperature. Nanoparticles were collected byultracentrifugation at 38,000 rpm for 1 hour. The bioactive agent, thethird component, tripolyphosphate and magnesium sulfate are wholly orsubstantially totally encapsulated in the core portion of thenanoparticles. Supernatants were discarded and nanoparticles wereresuspended in deionized water for freeze-drying preparation. Otheroperating conditions or other bioactive agent (such as protein, peptide,siRNA, growth factor, the one defined and disclosed herein, and thelike) may also apply.

Several conventional coating compounds that form a protective layer onparticles are used to physically coat or mix with the nanoparticlesbefore a freeze-drying process. The coating compounds may includetrehalose, mannitol, glycerol, and the like. Trehalose, also known asmycose, is an alpha-linked (disaccharide) sugar found extensively butnot abundantly in nature. It can be synthesized by fungi, plants andinvertebrate animals. It is implicated in anhydrobiosis—the ability ofplants and animals to withstand prolonged periods of desiccation. Thesugar is thought to form a gel phase as cells dehydrate, which preventsdisruption of internal cell organelles by effectively splinting them inposition. Rehydration then allows normal cellular activity to resumewithout the major, generally lethal damage, which would normally followa dehydration/rehydration cycle. Trehalose has the added advantage ofbeing an antioxidant.

Trehaloze has a chemical formula as C₁₂H₂₂O₁₁.2H₂O. It is listed as CASno. 99-20-7 and PubChem 7427. The molecular structure for trehalose isshown below.

Trehalose was first isolated from ergot of rye. Trehalose is anon-reducing sugar formed from two glucose units joined by a 1-1 alphabond giving it the name of α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside.The bonding makes trehalose very resistant to acid hydrolysis, andtherefore stable in solution at high temperatures even under acidicconditions. The bonding also keeps non-reducing sugars in closed-ringform, such that the aldehyde or ketone end-groups do not bind to thelysine or arginine residues of proteins (a process called glycation).Trehalose has about 45% the sweetness of sucrose. Trehalose is lesssoluble than sucrose, except at high temperatures (>80° C.). Trehaloseforms a rhomboid crystal as the dihydrate, and has 90% of the calorificcontent of sucrose in that form. Anhydrous forms of trehalose readilyregain moisture to form the dihydrate. Trehalose has also been used inat least one biopharmaceutical formulation, the monoclonal antibodytrastuzumab, marketed as Herceptin. It has a solubility of 68.9 g/100 gH₂O at 20° C.

Mannitol or hexan-1,2,3,4,5,6-hexyl (C₆H₈(OH)₆) is an osmotic diureticagent and a weak renal vasodilator. Chemically, mannitol is a sugaralcohol, or a polyol; it is similar to xylitol or sorbitol. However,mannitol has a tendency to lose a hydrogen ion in aqueous solutions,which causes the solution to become acidic. For this, it is not uncommonto add a substance to adjust its pH, such as sodium bicarbonate.Mannitol has a chemical formula as C₆H₁₄O₆. It is listed as CAS no.69-65-8 and PubChem 453. The molecular structure for mannitol is shownbelow.

Glycerol is a chemical compound with the formula HOCH₂CH(OH)CH₂OH. Thiscolorless, odorless, viscous liquid is widely used in pharmaceuticalformulations. Also commonly called glycerin or glycerine, it is a sugaralcohol and fittingly is sweet-tasting and of low toxicity. Glycerol hasthree hydrophilic alcoholic hydroxyl groups that are responsible for itssolubility in water and its hygroscopic nature. Glycerol has a chemicalformula as C₃H₅(OH)₃. It is listed as CAS no. 56-81-5. The molecularstructure for glycerol is shown below.

Example No. 19 Freeze-Drying Process for Nanoparticles

Nanoparticles (at 2.5% concentration) were mixed with solution from fourtypes of liquid at a 1:1 volume ratio for about 30 minutes until fullydispersed. The mixed particle-liquid was then freeze-dried under alyophilization condition, for example, at −80° C. and <25 mmHg pressurefor about 6 hours. The parameters in a selected lyophilization conditionmay vary slightly from the aforementioned numbers. The four types ofliquid used in the experiment include: (A) DI water; (B) trehalose; (C)mannitol; and (D) glycerol, whereas the concentration of the liquid (A)to liquid (C) in the solution was set at 2.5%, 5% and/or 10%. After afreeze-drying process, the mixed particle-liquid was rehydrated with DIwater at a 1:5 volume ratio to assess the integrity of nanoparticles ineach type of liquid. The results are shown in Table 6. By comparing theparticle size, polydispersity index and zeta-potential data, only thenanoparticles from the freeze-dried particle-trehalose runs (at 2.5%,5%, and 10% concentration level) show comparable properties as comparedto those of the before-lyophilization nanoparticles. Under the same dataanalysis, the nanoparticles from the freeze-dried particle-mannitol runs(at 2.5%, and 5% concentration level) show comparable properties ascompared to those of the before-lyophilization nanoparticles withrespect to size, Kcps, PI and Zeta potential. In one embodiment, asimilar experiment using a spray drying process (instead of afreeze-drying process) including trehalose and/or mannitol additive wasused to prepare the dried nanoparticles. The properties of thepost-drying nanoparticles are not statistically different from those ofthe pre-drying nanoparticles, demonstrating enablement of thefreeze-drying process and the spray drying process in preparingnanoparticle powders of the presently disclosed pharmaceuticalcomposition.

TABLE 6 Properties of nanoparticles before and after an exemplaryfreeze-drying process. A: DI Water A: DI water + NPs B: Trehalose C:Mannitol (volume 1:1), B: Trehalose + NPs (volume 1:1), C: Mannitol +NPs (volume 1:1), NPs solution freeze-dried freeze-dried freeze-driedConc. 2.50% Conc. Conc. 2.50% 5.00% 10.00% Conc. 2.50% 5.00% Size (nm)266 Size (nm) 9229.1 Size (nm) 302.4 316.7 318.9 Size (nm) 420.1 487.5Kcps 352.2 Kcps 465.3 Kcps 363.7 327.7 352.2 Kcps 305.4 303.7 PI 0.291PI 1 PI 0.361 0.311 0.266 PI 0.467 0.651 Zeta Potential 25.3 ZetaPotential Zeta Potential 25.6 24.6 24.7 Zeta Potential 24.4 25.3 D:Glycerol NPs solution D: Glycerol + NPs (volume 1:1), freeze-dried Conc.2.50% Conc. 2.50% 5.00% 10.00% Size (nm) 266 Size (nm) 6449.1 7790.31310.5 Kcps 352.2 Kcps 796.1 356.1 493.3 PI 0.291 PI 1 1 1 ZetaPotential 25.3 Zeta Potential

FIG. 16 shows an illustrative mechanism of nanoparticles released fromthe enteric-coated capsules. FIG. 16(A) shows the phase of nanoparticlesin the gastric cavity, wherein the freeze-dried nanoparticles 82 areencapsulated within an initial enteric coating or coated capsule 81.FIG. 16(B) shows a schematic of the nanoparticles during the phase ofentering small intestine, wherein the enteric coat and its associatedcapsule starts to dissolve 83 and a portion of nanoparticles 82 isreleased from the capsule and contacts fluid. FIG. 16(C) shows the phaseof nanoparticles in the intestinal tract, wherein the nanoparticlesrevert to a wet state having chitosan at its surface.

Example No. 20

Nanoparticles Encapsulated in an Alginate-Calcium Matrix

In an alternate embodiment, nanoparticles may be released fromalginate-calcium coating or matrix. Chemically, alginate is a linearcopolymer with homopolymeric blocks of (1-4)-linked β-D-mannuronate andits C-5 epimer α-L-guluronate residues, respectively, covalently linkedtogether in different sequences or blocks. Alginate has a molecularformula (C₆H₈O₆)_(n) and CAS number 9005-32-7. In preparation,nanoparticles are first suspended in a solution that contains calciumchloride, wherein the calcium ions are positively charged. With apipette, alginate having negatively charged carboxyl groups is slowlyadded to the calcium chloride solution. Under gentle stirring, thealginate-calcium starts to conjugate, gel, and coat on the nanoparticlesurface. In simulated oral administration of the alginate-calcium coatednanoparticles, nanoparticles start to separate from the coating whenthey enter the small intestines. In one embodiment, the alginate-calciumcoated nanoparticles are loaded in chewable tablets for oraladministration in an animal subject, including a human being.

Example No. 21 Freeze-Dried Nanoparticles in Animal Evaluation

In the in vivo study, rats as prepared and conditioned according toExample no. 12 were used in this evaluation. In the animal evaluationstudy, diabetic rats were fasting for 12 hours and subjected to threedifferent conditions: (a) oral deionized water (DI) administration asnegative control; (b) oral insulin-loaded lyophilized nanoparticlesadministration, whereas the nanoparticles have an insulin loadingcontent of 4.4% and an insulin loading efficiency of 48.6% and areloaded in a capsule with surface enteric coating; and (c) subcutaneous(SC) insulin injection at 5 U/kg as positive control. The blood glucoseconcentration from rat's tail was measured over the time in the study.

FIG. 19 shows glucose change (hypoglycemic index) versus time of the invivo animal study (n=5). The glucose change as a percentage of baselines for oral DI administration (control) over a time interval of 10hours appears relatively constant within the experimental measurementerror range. As anticipated, the glucose decrease for the SC insulininjection route appears in rat blood in the very early time interval andstarts to taper off after 2 hours in this exemplary study and ends atabout 6 hours. The most important observation of the study comes fromthe oral administration route with insulin-loaded lyophilized (namely,freeze-dried) nanoparticles. Nanoparticles of this example have insulinLC at 4.4%, whereas nanoparticles from Example no. 12 had insulin LC at14.1% in FIG. 15). With the same amount of nanoparticles in bothexamples, the insulin-feeding ratio of Example no. 21 to Example no. 12is about 1:3. In other words, the insulin fed to a rat in this studyfrom nanoparticles is about ⅓ of the insulin from nanoparticles fed torats in Example no. 12.

The blood glucose begins to decrease from the base line at about 3 hoursafter administration and sustains at a lower glucose level at more than10 hours into study. It implies that the current insulin-loadednanoparticles may modulate the glucose level in animals in a sustainedor prolonged effective mode. Some aspects of the invention provide amethod of treating diabetes of an animal subject, including a patient,comprising orally administering insulin-containing nanoparticles with adosage effective amount of the insulin to treat the diabetes, wherein atleast a portion of the nanoparticles comprises a positively chargedshell substrate and a negatively charged core substrate. In oneembodiment, the dosage effective amount of the insulin to treat thediabetes comprises an insulin amount of between about 15 units to 45units per kilogram body weight of the patient or an animal subject,preferably 20 to 40 units, and most preferably at about 25 to 35 unitsinsulin per kilogram body weight. In one embodiment, the lyophilizednanoparticles may be fed as is to an animal without being loaded in anenterically coated capsule.

It is known that Zn (zinc) is usually added in the biosynthesis andstorage of insulin. FIGS. 17 and 18 show a schematic of insulinconjugated with a polyanionic compound (i.e., γ-PGA in this case) via Znand thus increase its loading efficiency and loading content in thenanoparticles of the present invention. It is further demonstrated thatZn may complex with the histidine and glutamic acid residues in insulinto increase the insulin stability and enhance controlled releasecapability or sustained therapy. Some aspects of the invention relate toa nanoparticle characterized by enhancing intestinal or brain bloodparacellular transport, the nanoparticle comprising a first component ofat least one bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein astabilizer is added to complex the at least one bioactive agent to thenegatively charged third component. In one embodiment, the stabilizer iszinc or calcium.

Example No. 22 Nanoparticles with Enhanced Insulin Loading

In a co-pending application, U.S. patent application Ser. No. 11/881,185filed Jul. 26, 2007, entire contents of which are incorporated herein byreference, it is disclosed that a novel nanoparticle may comprise ashell substrate of chitosan and a core substrate consisting of at leastone bioactive agent, MgSO₄, TPP, and a negatively charged substrate thatis neutralized with chitosan in the core. FIG. 20 shows insulin-loadednanoparticles with a core composition comprised of γ-PGA, MgSO₄, sodiumtripolyphosphate (TPP), and insulin. Nanoparticles were obtained uponaddition of core component, using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a CS aqueous solution (pH 6.0,10 ml) at certain concentrations under magnetic stirring at roomtemperature. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. Supernatants were discarded and nanoparticleswere resuspended in deionized water for further studies. In oneembodiment, nanoparticles are encapsulated in a gelcap or arelyophilized before being loaded in a gelcap or in a tablet. The sodiumtripolyphosphate has a chemical formula of Na₅P₃O₁₀ as shown below:

In the example, the core composition may be varied and evaluated with apreferred composition of 2 ml γ-PGA aqueous solution at pH 7.4 plusinsulin, MgSO₄ and TPP, resulting in a ratio ofCS:γ-PGA:TPP:MgSO4:insulin=6.0:1.0:1.0:2.0:0.05. Thus, the nanoparticlesshow characteristics with chitosan shell and a core compositionconsisted of γ-PGA, MgSO₄, TPP, chitosan, and insulin and have anaverage loading efficiency of 72.8% insulin and an average loadingcontent of 21.6% insulin.

In the enhanced drug loading of the present example, there provides twoor more distinct ionic crosslink mechanisms. In one embodiment, thenanoparticles of the present invention may have a structure or matrix ofinterpenetrated ionic-crosslinks (that is, elongate ionic-crosslinkchains) including a first ionic-crosslink chain of NH₃ ⁺ of CS with COO⁻of γ-PGA, a second ionic-crosslink chain of NH₃ ⁺ of CS with SO₄ ²⁻ ofMgSO₄, a third ionic-crosslink chain of Mg²⁺ of MgSO₄ with COO⁻ ofγ-PGA, and/or a fourth ionic-crosslink chain of Na₃P₃O₁₀ ²⁻ of TPP withNH₃ ⁺ of CS or Mg²⁺ of MgSO₄.

Some aspects of the invention relate to a nanoparticle composition fororal administration with the insulin loading efficiency and content athigher than 45% and 14% (preferably up to about 73% and 22%),respectively. The prepared nanoparticles (NPs) are stable in the rangeof pH 2.0 to 7.1. This broad range is to maintain the chitosan-shellednanoparticle transiently stable in most of the intestine region(including duodenum, jejunum, and ileum) for enhanced membraneadsorption and paracellular permeability of active ingredient (forexample, insulin, exenatide or pramlintide). Some aspects of theinvention provide a chitosan-shelled nanoparticle with a corecomposition comprised of γ-PGA, MgSO₄, TPP, chitosan, and at least onebioactive agent, such as insulin, exenatide or pramlintide for treatmentof diabetes. In an alternate embodiment, some aspects of the inventionprovide a chitosan-shelled nanoparticle with a core compositionconsisted of γ-PGA, MgSO₄, TPP, chitosan, and at least one bioactiveagent. In one embodiment, negatively charged γ-PGA may conveniently besubstituted in preparation by another negatively charge substrate, suchas heparin. In an experiment following the experimental conditions ofExample no. 22 by substituting insulin with exenatide, chitosan-shellednanoparticles with a core composition comprised of γ-PGA, MgSO₄, TPP,chitosan, and exenatide have been prepared that exhibit similar physicaland mechanical properties as compared to the ones with insulin.

FIG. 21 shows an in vivo subcutaneous study using insulin injectablesand insulin-containing nanoparticles. The insulin-containingnanoparticles exhibit different pharmacodynamics and/or pharmacokineticsin a sustained releasing manner. Some aspects of the invention relate toa pharmaceutical composition of nanoparticles for subcutaneous or bloodvessel administration in an animal subject, including a patient, thenanoparticles comprising a shell portion that is dominated by positivelycharged chitosan, a core portion that contains negatively chargedsubstrate, wherein the negatively charged substrate is at leastpartially neutralized with a portion of the positively charged chitosanin the core portion, and at least one bioactive agent loaded within thenanoparticles.

Some aspects of the invention relate to a method of delivering abioactive agent to blood circulation in an animal subject, including apatient, comprising: (a) providing nanoparticles according to apreferred embodiment of the pharmaceutical composition of the presentinvention, wherein the nanoparticles are formed via a simple and mildionic-gelation method; (b) administering the nanoparticles orally towardthe intestine of the patient or the animal subject via stomach; (c)urging the nanoparticles to be absorbed onto a surface of an epithelialmembrane of the intestine via muco-adhesive chitosan-shellednanoparticles; (d) permeating bioactive agent to pass through anepithelial barrier of the intestine; and (e) releasing the bioactiveagent into the blood circulation. In one embodiment, the bioactive agentis selected from the group consisting of exenatide, pramlintide,insulin, insulin analog, and combinations thereof. In anotherembodiment, the bioactive agent permeates through the tight junctions ofthe epithelial membrane when chitosan-shelled nanoparticles break up andrelease the bioactive agent at vicinity of the tight junctions.

Some aspects of the invention relate to a method for inducing aredistribution of tight junction's ZO-1 protein, leading totranslocation of the ZO-1 protein to cytoskeleton that accompaniesincreased paracellular transport in an animal subject, including apatient, the method comprising administering into the animal subject,including a patient, bioactive nanoparticles with a dosage effective toinduce the redistribution, wherein the bioactive nanoparticles comprisea shell substrate of chitosan and a core substrate that comprisespoly(glutamic acid) and the bioactive agent that is selected from thegroup consisting of exenatide, pramlintide, insulin, insulin analog, andcombinations thereof.

Example No. 23 Nanoparticles Embedded in a Jerry

Nanoparticles (also known as bioactive nanoparticles because thenanoparticles contain at least one bioactive agent) of the presentinvention can be embedded in a jerry (gelatin) for feeding an animalsubject, including a baby or human being. In one example, thenanoparticles from Example no. 22 show some characteristics as disclosedherein in the present patent application. The nanoparticles showcharacteristics with chitosan-dominated shell and a core compositionconsisted of γ-PGA, MgSO₄, TPP, chitosan, and insulin and have anaverage loading efficiency of 72.8% insulin and an average loadingcontent of 21.6% insulin. In one embodiment, a pharmaceuticalcomposition of nanoparticles for oral administration in an animalsubject, the nanoparticles comprising a shell portion that is dominatedby positively charged chitosan, a core portion that contains negativelycharged substrate, wherein the negatively charged substrate is at leastpartially neutralized (preferably substantially neutralized, and mostpreferably fully neutralized) via electron-interaction with a portion ofthe positively charged chitosan in the core portion, and at least onebioactive agent loaded within the nanoparticles. In another example, 10mg of the nanoparticles from Example no. 22 is dispersed in 100 mg ofliquid jerry at about 37° C., optionally with continuous stirring. Thenanoparticles-containing liquid jerry is then cooled to about 4° C. tobecome a gel form. The gel jerry is, optionally fragmented, then fed tothe animal subject. For example, a flavored gel jerry havingnanoparticles of the present invention is prepared and configured forfeeding to a diabetic patient or animal subject.

In one embodiment, nanoparticles are first loaded within biodegradablecationic polymers and then blended with other nutrient/filleringredients in a chewable formulation as animal food. Cationic polymersare positively charged polymers. Their positive charges prevent theformation of coiled polymers. This allows them to contribute more toviscosity in their stretched state, because the stretched-out polymertakes up more space than a coiled polymer and thus resists the flow ofsolvent molecules around it. Some cationic polymer formulas includevinylpyrrolidone, methacrylamide, hydrogel N-vinylimidazole, and acopolymer thereof.

Example No. 24 Nanoparticles Loaded in Temperature-Responsive Hydrogels

Nanoparticles of the present invention can be embedded in or coated withtemperature-responsive hydrogels for feeding an animal subject. In aco-pending application, U.S. patent application Ser. No. 11/256,729filed Oct. 24, 2005, it is disclosed a thermo-responsive methylcellulose(MC) or poly(N-isopropylacrylamide) hydrogel. For illustration purposes,aqueous MC solutions or poly(N-isopropylacrylamide) hydrogel solutionsundergo a sol-gel reversible transition upon heating or cooling. In thesolution state at lower temperatures, MC molecules are hydrated andthere is little polymer-polymer interaction other than simpleentanglements. As temperature is increased, aqueous MC solutions absorbenergy and gradually lose their water of hydration. Eventually, apolymer-polymer association takes place, due to hydrophobicinteractions, causing cloudiness in solution and subsequently forming aninfinite gel-network structure at the gelation temperature.

MC (with a viscosity of 3,000-5,500 cps for a 2% by w/v aqueous solutionat 20° C.) was obtained from Fluka (64630 Methocel® MC, Buchs,Switzerland). Aqueous MC solutions in different concentrations (1%, 2%,3%, or 4% by w/v) were prepared by dispersing the weighed MC powders inheated water with the addition of distinct salts (NaCl, Na₂SO₄, Na₃PO₄)or in phosphate buffered saline (PBS) in varying concentrations at 50°C. The osmolalities of the prepared aqueous MC solutions were thenmeasured using an osmometer (Model 3300, Advanced Instruments, Inc.,Norwood, Mass., USA). The prepared MC has a gelation temperature atabout 37° C.

In one example, 10 mg of the nanoparticles from Example no. 22 isdispersed in 100 mg of liquid MC at about 20° C., optionally withcontinuous stirring. The nanoparticles-containing liquid MC is thenheated to about 37° C. to become a gel form. The gel MC is, optionallyfragmented, then used for feeding a patient or an animal subject.

Example No. 25 Nanoparticles Loaded within Hydrogels

Nanoparticles of the present invention are generally stable in anenvironment of a pH range between about 2.5 to about 7.0. Nanoparticlescan be embedded in, loaded in or coated with gels (such as hydrogels,organogels, or xerogels) that are stable in the range of about 1.0 toabout 5.5 as food-like medicine for feeding an animal subject. There areseveral possible mechanisms that could lead to gel formation from aliquid hydrogel, such as solvent exchange, UV-irradiation, ioniccross-linkage, pH change, and temperature modulation. In one embodiment,a preferred hydrogel is stable in a solid gel form at a pH lower thanabout 5.5, preferably lower than about 2.5 (or between about 1.0 and2.5) so the hydrogel-coated nanoparticles are relatively stable whenpassing the stomach chamber of an animal subject. In another embodiment,the above-prepared hydrogel-coated nanoparticles are formulated intochewable animal food.

A gel is an apparently solid, jelly-like material formed from acolloidal solution. Hydrogel is a network of polymer chains that arewater-insoluble, sometimes found as a colloidal gel in which water isthe dispersion medium. Hydrogels are superabsorbent (they can containover 99% water) natural or synthetic polymers. Hydrogels possess also adegree of flexibility very similar to natural tissue, due to theirsignificant water content. An organogel is a non-crystalline, non-glassythermoreversible solid material composed of a liquid organic phaseentrapped in a structuring network. The liquid can be e.g. an organicsolvent, a mineral oil or a vegetable oil. The solubility and particledimensions of the structure are important characteristics for theelastic properties and firmness of the organogel. Often, these systemsare based on self-assembly of the structuring molecules. A xerogel is asolid formed from a gel by drying with unhindered shrinkage. Xerogelsusually retain high porosity (25%) and enormous surface area (150-900m²/g), along with very small pore size (1-10 nm). When solvent removaloccurs under hypercritical (supercritical) conditions, the network doesnot shrink and a highly porous, low-density material known as an aerogelis produced.

Example No. 26 Chewable Formulation Having Nanoparticles

As disclosed herein, nanoparticles of the present invention that containat least one bioactive agent can be orally delivered to an animalsubject, including a person, via direct oral administration or beingassociated with a drug delivery carrier for oral administration. Thedrug delivery carrier may comprise a form of encapsulation (such as in acapsule, a chewable capsule, a softgel capsule, a soft chewable shell,and the like), entrapment or chewable (such as in tablets, soft chewabletablets, pills, lyophilized nanoparticles, chewable substrate, chewablegels, and the like), coating (such as in alginate-calcium coatednanoparticles and the like), or the combinations thereof (such as thealginate-calcium coated nanoparticles loaded in chewable tablets and thelike).

U.S. Pat. No. 4,882,152, entire contents of which are incorporatedherein by reference, discloses an active ingredient pre-coated withglycerides, lecithin, polyoxyalkylenes, or polyalkylene glycols, andmixed into a binder comprising a gelatin, a sweetener, glycerin, andwater.

U.S. Pat. No. 7,029,699, entire contents of which are incorporatedherein by reference, discloses a process for making a compressed,chewable tablet containing at least one active ingredient, awater-disintegratable, compressible carbohydrate and a binder.

U.S. Pat. No. 5,200,191, entire contents of which are incorporatedherein by reference, discloses a softgel manufacturing process. Aftersoftgels are encapsulated and dried in a drying tunnel, the resultingsoftgels are subjected to a further stress-relieving step. During thestress-relieving step, the temperature and humidity conditions in thedrying tunnel are heightened. By utilizing the stress-relieving step,the volume and number of dimples and bubbles in the softgels arereduced, and dimensional uniformity is maximized.

U.S. Pat. No. 5,380,535, entire contents of which are incorporatedherein by reference, discloses chewable drug-delivery compositions andmethods of preparation. A non-aqueous, chewable composition for oraldelivery of unpalatable drugs is provided along with preparative methodstherefore. The composition contains the drug intimately dispersed ordissolved in a pharmaceutically-acceptable lipid that is solid at roomtemperatures. The composition also has a matrix that contains agranulating agent for the total composition and a rapid dispersal agentand optionally additives such as buffering agents, flavoring agents,surfactants, and the like.

Chewable tablets, regardless their geometry, represent a particular formof oral dosage; they are intended to be chewed in the mouth by theanimal subject. The chewable formulation of the nanoparticles of thepresent invention is preferred with no or little heat. The chewableformulation of the nanoparticles of the present invention is alsopreferred with no or little pressure. To achieve acceptable stabilityand quality as well as good taste and mouth feel in a chewableformulation of the oral nanoparticle compositions of this inventionseveral considerations are important. These considerations include theamount of active substance per tablet, the flavoring agent employed, thedegree of compressibility of the tablet and the organoleptic propertiesof the composition. Excipients when added to chewable tablets must notonly be inert in respect of the active, preferably they provide pleasantmouth-feel and/or prevent tooth-packing, grittiness, and the like,without imparting any unpleasant characteristics to the tablets as theyare chewed. The composition of the present invention may also have amatrix that contains a granulating agent for the total composition and arapid dispersal agent and optionally additives such as buffering agents,flavoring agents, surfactants, and the like.

In one embodiment, a chewable composition of the present invention maybe in the form of a tablet comprising a core containing bioactivenanoparticles and an outer layer of chewable base wrapping the core. Thechewable tablet of the present invention may be prepared by thefollowing exemplary process comprising the steps of: (a) preparing anouter layer of chewable base and a core of jelly or chewable base,respectively; (b) mixing bioactive nanoparticles of the presentinvention with the jelly or chewable base of the core at roomtemperature (referred to as “core substrate”); (c) loading the chewablebase and the core substrate into two extruding machines (for example,the one disclosed in Korean Patent application No. 98-30511),respectively, and then each of the outer layer and the core substrate isextruded at the same time; and (d) forming a chewable tablet by cuttingthe above co-extruded material.

In one embodiment, a chewable composition of the present invention maybe in the form of a pellet comprising bioactive nanoparticles that areblended or dispersed in filler substance (i.e., filler base material).The chewable pellet of the present invention may be prepared by thefollowing exemplary process. A mix was prepared by mixing thenanoparticles (preferably in a freeze-dried powder form or othersuitable form) and filler substance together. The mix was fed into anextruder having an inlet temperature of about room temperature and fedalong a transfer zone. Water was then added (if needed) to the mix inthe mixing zone to provide the moist mix. In the case of wetnanoparticles in the mix, water may not be needed. The paddles in themixing zone were adjusted accordingly. The extrudate can be cut and airdried as animal foodstuffs containing bioactive agents or can be fedinto a pellet-forming machine to form pellets as animal foodstuffshaving bioactive agents. Cold extrusion is done at room temperature ornear room temperature. The advantages of this over hot extrusion are thelack of oxidation, higher strength due to cold working, closertolerances, good surface finish, and/or fast extrusion speeds.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims. Many modifications and variations are possible inlight of the above disclosure.

1. A chewable composition for feeding an animal subject comprising afiller substance and nanoparticles that are blended with said fillersubstance, said nanoparticles comprising a shell portion that isdominated by positively charged chitosan, a core portion that containsnegatively charged substrate, wherein said negatively charged substrateis at least partially neutralized with a portion of said positivelycharged chitosan in the core portion, and at least one bioactive agentloaded within said nanoparticles.
 2. The chewable composition of claim1, wherein said chewable is configured for oral administration in ananimal subject.
 3. The chewable composition of claim 1, wherein saidsubstrate in the core portion is PGA or heparin.
 4. The chewablecomposition of claim 1, wherein said nanoparticles are formed via asimple and mild ionic-gelation method.
 5. The chewable composition ofclaim 1, wherein said nanoparticles further comprise magnesium sulfateand TPP in the core portion.
 6. The chewable composition of claim 1,wherein said chewable in is the form of tablets, soft chewable tablets,or pills.
 7. The chewable composition of claim 1, wherein said chitosanof the nanoparticles is trimethyl chitosan.
 8. The chewable compositionof claim 1, wherein said bioactive agent is insulin or insulin analog.9. The chewable composition of claim 1, wherein said nanoparticles arefreeze-fried.
 10. The chewable composition of claim 1, wherein saidbioactive agent is exenatide or pramlintide.
 11. The chewablecomposition of claim 1, wherein said bioactive agent is protein orpeptide.
 12. The chewable composition of claim 1, wherein saidnanoparticles are embedded in a jerry.
 13. The chewable composition ofclaim 1, wherein said bioactive agent is calcitonin.
 14. The chewablecomposition of claim 1, wherein said bioactive agent is vancomycin. 15.The chewable composition of claim 1, wherein said nanoparticles arecoated with hydrogels.
 16. The chewable composition of claim 1, whereinthe filler substance comprises excipients.
 17. The chewable compositionof claim 1, wherein said nanoparticles are coated with analginate-calcium matrix.
 18. The chewable composition of claim 1,wherein said bioactive agent is growth hormone.
 19. The chewablecomposition of claim 1, wherein said nanoparticles further comprise anabsorption enhancer.
 20. The chewable composition of claim 1, whereinsaid nanoparticles are loaded in organogels or xerogels.